<span style="font-size: 11pt; line-height: 115%; font-family: Arial, sans-serif; color: rgb(41, 43, 49); background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;">We are very grateful to Gudrun Wolter and &#196;nne Michaelis for excellent technical assistance. We would like to thank all the members of the Giavalisco lab for their help. We are thankful to the Max Planck Society for funding the research and FAPESP for the fellowship of L A Giraldi

1. Pre-cultures of Chlamydomonas reinhardtii
<ol>
	<li>Prepare the pre-cultures of Chlamydomonas reinhardtii (strain wild type CC-1690 mt+) by transferring cells from solid plates of TAP medium to Erlenmeyer flasks with 200 mL of HSM medium12.</li>
	<li>Grow the pre-cultures on a rotatory shaker at 24 &#176;C at 100 &#181;moles&#183;m-2&#183;s-1 continuous light until the cell density reaches ~ 2 x 106 cells/mL.</li>
</ol>
2. Synchronization of Chlamydomonas liquid cultures in fermenters
NOTE: The parameters presented in this protocol, such as temperature, light and CO2, are specific to synchronization of the strain CC-1690 mt+. In order to develop a synchronized culture using another strain, it is necessary to test the optimal conditions.
<ol>
	<li>Transfer the pre-culture to a fermenter system (see Supplementary Figure 1 for the custom-made fermenter design), connected to a recirculating cooler to maintain the temperature, with bubbling of filtered CO2 (2%, v/v) and stirred with a magnetic stir bar at the bottom of the fermenter with constant agitation.</li>
	<li>To initiate the synchronization, set the fermenter at light-dark regime of 12:12 h, under 34 &#176;C and 200 &#181;moles&#183;m-2&#183;s-1 light and ensure it contains 500 mL of culture in HSM medium with a cell density of 1 x 106 cells/mL.</li>
	<li>At the end of 24 h cycle, re-dilute the culture to 1 x 106 cells/mL with fresh HSM medium with minimal perturbation by using a tube system in combination with peristaltic pumps, which allows to first pump a distinct amount of culture out and afterwards pump in autoclave-sterilized medium. 
	NOTE: As an alternate to peristaltic pumps, inoculating syringes can be used to withdraw the culture, while the required volume of fresh medium can be added directly into the fermenter under sterile conditions.</li>
	<li>Repeat step 2.3 three times to obtain the synchronized cultures on 4th day of inoculation.</li>
	<li>In order to validate synchrony of cells, harvest samples at an interval of every 2 h and measure the cell density and cell volume using a cell counter and size analyzer (see Table of Materials). Depending on the cell density, dilute the samples between 1:10 to 1:100 and measure in a size range of 30 &#8211; 1900 &#181;m3.</li>
</ol>
3. Harvesting Chlamydomonas cells
<ol>
	<li>Label 15 mL conical centrifuge tubes and prepare a dewar filled with liquid nitrogen.</li>
	<li>Harvest samples using a syringe (50 cm3) through the inner glass tube of the fermenter. Distribute the sample into conical centrifuge tubes which should contain 10-15 x 106 cells. Note the volume transferred to each tube and measure the cell density and the cell size of each time point using cell counter and size analyzer (see Table of Materials)</li>
	<li>Pellet the cells by centrifugation for 5 min at 4000 x g. Then discard the supernatant, freeze the pellets in liquid nitrogen and later store at -80 &#176;C.</li>
</ol>
4. Preparation of extraction buffers and extraction chlorophyll, lipids and metabolites
CAUTION: Methanol (MeOH) and MTBE are flammable and can cause irritation of respiratory tract, eye or skin on prolonged exposure and/or contact. Please handle them carefully only in a fume hood and use the appropriate safety procedures during the extraction (lab coat, safety glasses, gloves, etc.).
<ol>
	<li>Extraction Buffer 1
	<ol>
		<li>In a 100 mL volumetric flask, add 75 mL of MTBE, 25 mL of MeOH (UHPLC grade) and homogenize (3:1, vol/vol).</li>
		<li>Add the following internal standards solution: 50 &#181;L of corticosterone (1 mg/mL in MeOH), 50 &#181;L 1,2-diheptadecanoyl-SN-glycero-3-phosphocholine (17:0 PC) (1 mg/mL in chloroform HPLC grade), 25 &#181;L of ampicillin (1 mg/mL in water UHPLC grade) and 50 &#181;L of D-Sorbitol-1-13C (1 mg/mL in water UHPLC grade).</li>
		<li>Transfer the extraction buffer to a clean glass bottle and pre-cool the solution at -20 &#176;C, 1 h before use. Use freshly prepared solution for the extractions. This solution can be stored at 4 &#176;C for up to 2 weeks.</li>
	</ol>
	</li>
	<li>Extraction Buffer 2
	<ol>
		<li>In a 100 mL volumetric flask, add 75 mL of water UHPLC grade and 25 mL of MeOH UHPLC grade and homogenize.</li>
		<li>Transfer the extraction buffer to a clean glass bottle. Use fresh solution for the extractions. This solution can be stored at room temperature for several weeks.</li>
	</ol>
	</li>
</ol>
5. Extraction of chlorophyll, lipids and metabolites
<ol>
	<li>Arrange the tubes, with the cell pellet (containing 10-15 x 106 cells), in liquid nitrogen.</li>
	<li>Resuspend the cell pellet in each tube with 1 mL of pre-cooled (-20 &#176;C) Extraction Buffer 1. 
	NOTE: Perform this step quickly to avoid evaporation of low viscosity extraction buffer. After adding the Extraction Buffer 1, maintain the tubes at room temperature.</li>
	<li>Vortex until the cells are well homogenized within the extraction mixture and aliquot the mixture into 2 mL microcentrifuge tube.</li>
	<li>Sonicate the cultures using sonication bath (see Table of Materials) in ice cooled water for 10 min.</li>
	<li>Incubate all the samples on an orbital shaker at 1000 rpm for 60 min at 4 &#176;C.</li>
	<li>To induce the phase separation, add 650 &#181;L of Extraction Buffer 2.</li>
	<li>Vortex briefly followed by centrifugation at 20000 x g for 5 min at 4 &#176;C. 
	NOTE: After this step, there is a separation of liquid phases and a solid pellet in the bottom of the tube. Handle the tubes with care to avoid mixing of the two liquid phases. The top MTBE-phase contains lipids and chlorophyll, while the lower phase contains the polar and semi-polar metabolites. The precipitated pellet at the bottom contains proteins, starch and other insoluble molecules.</li>
</ol>
6. Aliquoting the fractions
<ol>
	<li>Transfer 500 &#181;L of upper MTBE-phase (lipids) into a labelled 1.5 mL tube. Dry the samples using a vacuum concentrator (see Table of Materials) and store at -80 &#176;C until measurement.</li>
	<li>Remove the remaining MTBE-phase using a 200 &#956;L pipette.</li>
	<li>Transfer 650 &#181;L of the lower phase (polar and semi-polar metabolites) to a new labeled tube. Dry the samples using a vacuum concentrator (see Table of Materials) and store at -80 &#176;C until measurement.</li>
	<li>Remove the remaining lower phase by pipetting off the excess volume.</li>
	<li>Freeze the solid pellet in liquid nitrogen and store at -80 &#176;C until further extraction.</li>
</ol>
7. Determination of polar metabolites (primary metabolites)
<ol>
	<li>Resuspend the dried pellet of the polar phase (step 6.3) in methoxyamine-hydrochloride/pyridine solution for methoxymization of carbonyl groups.</li>
	<li>Heat the samples at 37 &#176;C for 90 min.</li>
	<li>Derivatize the samples with N-methyl-N-trimethylsilyltrifloracetamide (MSTFA) for 30 min at 37 &#176;C as described previously13.</li>
	<li>Use gas chromatography coupled to time-of-flight mass spectrometry (GC-TOF-MS) to analyze the primary metabolites. The gradient parameters used were according to the previously described protocol14.</li>
</ol>
8. Determination of non-polar metabolites (Lipids)
<ol>
	<li>Re-suspend the dried pellet of non-polar phase (step 6.1) in a mixture of acetonitrile:isopropanol (7:3, v:v).</li>
	<li>Centrifuge at 20000 x g and separate on a reverse phase C8 column (100 mm&#215;2.1 mm&#215;1.7 &#956;m particles), using a UPLC system (see Table of Materials). The two mobile phases were water with 1% 1 M ammonium acetate, 0.1% acetic acid (Buffer A), and acetonitrile:isopropanol (7:3) containing 1% 1 M ammonium acetate, 0.1% acetic acid (Buffer B).</li>
	<li>Use the gradient parameters according to the previously described protocol14.</li>
</ol>
9. Determination of chlorophyll content
<ol>
	<li>Mix 100 &#181;L of the MTBE-phase (step 6.1) with 900 &#181;L of 90% methanol for a method blank as well as the experimental samples.</li>
	<li>Measure the absorbance using spectrophotometer at a wavelength of 665 nm and 652 nm to distinguish between chlorophyll a and chlorophyll b. 
	NOTE: The absorption should range between 0.1 and 1 for valid concentration calculation. Dilute the samples, if necessary.</li>
	<li>Calculate the chlorophyll a and b content, and also the total chlorophyll content according to the following formulas. 
	Chla = 16.82A665 &#8211; 9.28A652 
	Chlb = 36.92A652 &#8211; 16.54A665 
	Chla=b = 0.28A665 + 27.64A652 
	NOTE: The formulas were described by Bar-Nun and Ohad15.</li>
</ol>
10. Extraction and determination of the protein content, digestion and analysis
NOTE: To resuspend the protein, pellet urea/thiourea buffer (6 M urea, 2 M thioureaand protease and phosphatase inhibitors) was used with modifications in the protocol previously described16. However, any buffer of choice can be used for resuspension of proteins.
<ol>
	<li>Prepare the protein buffer using the following concentrations: 6 M of urea and 2 M of thiourea.</li>
	<li>Dissolve the pellet (step 6.5) in 200 &#181;L of protein buffer (10.1).</li>
	<li>Incubate the samples at room temperature for 30 min, followed by centrifugation at 20000 x g for 5 min.</li>
	<li>Transfer the supernatant, which contains the proteins, to a new tube.</li>
	<li>Determine the protein concentration by Bradford assay17.</li>
	<li>Digest 50 &#181;g of protein in-solution with a protocol of choice. Here, use the following protocol.
	<ol>
		<li>Reduce 50 &#181;g of protein samples using 5 mM DTT for 30 min followed by alkylation using 10 mM iodoacetamide for 30 min at room temperature in dark.</li>
		<li>Add Trypsin/Lys-C Mix at a 25:1 protein:protease ratio (w/w), mix and incubate for 3 h at 37 &#176;C.</li>
		<li>Dilute the samples six folds using 50 mM TrisHCl (pH 8) and Incubate overnight at 37 &#176;C.</li>
		<li>Terminate digestion by adding trifluoroacetic acid (TFA) to a final concentration of 0.5&#8211;1%.</li>
	</ol>
	</li>
	<li>After digestion, perform desalting of the peptides prior to mass spectrometry using C18 stage tips and elute the digested peptides18.</li>
	<li>Concentrate the samples to near dryness, leaving 2-5 &#181;L of solution in a vacuum concentrator (see Table of Materials) without heating.</li>
	<li>Resuspend the samples in loading buffer (5% acetonitrile, 0.5% formic acid) and analyze the peptide mixtures by LC-MS/MS using a high-resolution mass spectrometer (see Table of Materials) connected to a nano-UPLC system.</li>
	<li>Separate the peptides using UPLC system (see Table of Materials) on a 20 cm reverse phase charged surface hybrid (CSH) column (see Table of Materials) with an inner diameter of 75 &#956;m and a particle size of 1.7 &#956;m.</li>
	<li>Load 4 &#181;L of sample and run through 90 min gradient at a flow rate of 300 nL/min.</li>
	<li>Set the gradient. Use partial loop-offline settings with isocratic gradient set at 3% of buffer B (99.9% acetonitrile + 0.1% formic acid) held for 14 min before the loop is shifted to online position with the column, thereafter the gradient is increased linearly for 50 min, until 20% buffer B is reached.</li>
	<li>Within the next 15 min, increase the concentration of buffer B to 30%. Then in the following 15 min, increase buffer B to 40%, before reaching 90% of Buffer B after another 4 min. Perform the washing step, which is required to clean the column, at 400 nL/min and hold for additional 10 min (see Table 1 for detailed gradient conditions).</li>
	<li>Finally, set back the system to a flow rate of 300 nL/min and a concentration of 3% buffer B within 1 min. Equilibrate the column for 15 min before the next sample is injected. 
	NOTE: A complete list of identified proteins after LCMS/MS analysis is presented in Table 2.</li>
</ol>
11. Extraction and determination of starch content
NOTE: For determination of total protein and starch content, the solid pellet was extracted in a two-step procedure as described previously10.
<ol>
	<li>Add 500 &#181;L of 80% (v/v) ethanol to the cell pellet (step 6.5) and incubate for 10 min at 80 &#176;C.</li>
	<li>Centrifuge at 4000 x g for 10 min at room temperature. Then dissolve the pellet in 250 &#181;L of sterile water followed by the addition of 250 &#181;L of 100 mM sodium acetate.</li>
	<li>Hydrolyze the starch by heating for 3 h at 99 &#176;C. Digest the dissolved starch into glucose monomers overnight by adding an enzyme mix of &#945;-amylase (4.2 units per sample) and &#945;-amyloglucosidase (10 units per sample). Incubate the tubes at 37 &#176;C overnight. 
	NOTE: After incubation, samples can be frozen at -20 &#176;C for few days, or at -80 &#176;C for several months, prior to glucose measurements.</li>
	<li>Centrifuge the digested extract at 20000 x g for 5 min and collect the supernatant. Dissolve the supernatant (with the starch-derived glucose monomers) in 100 mM of HEPES-buffer (pH 7) that contains 5 mM MgCl2, 60 mg/mL ATP, 36 mg/mL NADP and glucose-6-phosphate-dehydrogenase (1 unit per sample).</li>
	<li>In order to analyze the starch content, first measure the baseline absorbance at 340 nm. Then add hexokinase (1 unit per sample) to start the reaction. Finally measure the increase of NADPH+H+ (indicating level of starch digested to glucose) at 340 nm with a 96-well plate reader. 
	NOTE: The difference of the maximum value at 340 nm (should not exceed the linear range of 1) and the baseline is equivalent to the glucose concentration of the digested starch. A glucose curve should be made to determine the glucose concentration in nmol of glucose per mL.</li>
</ol>

<ol>
	<li>Spolaore, P., Joannis-Cassan, C., Duran, E., Isambert, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Commercial+applications+of+microalgae.">Commercial applications of microalgae.</a> Journal of Bioscience and Bioengineering. 101 (2), 87-96 (2006).</li><li>Georgianna, D. R., Mayfield, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploiting+diversity+and+synthetic+biology+for+the+production+of+algal+biofuels.">Exploiting diversity and synthetic biology for the production of algal biofuels.</a> Nature. 488 (7411), 329-335 (2012).</li><li>Pulz, O., Gross, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Valuable+products+from+biotechnology+of+microalgae.">Valuable products from biotechnology of microalgae.</a> Applied Microbiology and Biotechnology. 65 (6), 635-648 (2004).</li><li>Perez-Garcia, O., Escalante, F. M., de-Bashan, L. E., Bashan, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterotrophic+cultures+of+microalgae:+metabolism+and+potential+products.">Heterotrophic cultures of microalgae: metabolism and potential products.</a> Water Research. 45 (1), 11-36 (2011).</li><li>Mukhopadhyay, A., Redding, A. M., Rutherford, B. J., Keasling, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Importance+of+systems+biology+in+engineering+microbes+for+biofuel+production.">Importance of systems biology in engineering microbes for biofuel production.</a> Current Opinion in Biotechnology. 19 (3), 228-234 (2008).</li><li>Chang, R. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+network+reconstruction+of+Chlamydomonas+offers+insight+into+light-driven+algal+metabolism.">Metabolic network reconstruction of Chlamydomonas offers insight into light-driven algal metabolism.</a> Molecular Systems Biology. 7, 518 (2011).</li><li>Dal'Molin, C. G., Quek, L. E., Palfreyman, R. W., Nielsen, L. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AlgaGEM--a+genome-scale+metabolic+reconstruction+of+algae+based+on+the+Chlamydomonas+reinhardtii+genome.">AlgaGEM--a genome-scale metabolic reconstruction of algae based on the Chlamydomonas reinhardtii genome.</a> Bmc Genomics. 12 Suppl 4, S5 (2011).</li><li>Zones, J. M., Blaby, I. K., Merchant, S. S., Umen, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-Resolution+Profiling+of+a+Synchronized+Diurnal+Transcriptome+from+Chlamydomonas+reinhardtii+Reveals+Continuous+Cell+and+Metabolic+Differentiation.">High-Resolution Profiling of a Synchronized Diurnal Transcriptome from Chlamydomonas reinhardtii Reveals Continuous Cell and Metabolic Differentiation.</a> Plant Cell. 27 (10), 2743-2769 (2015).</li><li>Willamme, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolomic+analysis+of+the+green+microalga+Chlamydomonas+reinhardtii+cultivated+under+day/night+conditions.">Metabolomic analysis of the green microalga Chlamydomonas reinhardtii cultivated under day/night conditions.</a> Journal of Biotechnology. 215, 20-26 (2015).</li><li>Juppner, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+lipids+and+metabolites+during+the+cell+cycle+of+Chlamydomonas+reinhardtii.">Dynamics of lipids and metabolites during the cell cycle of Chlamydomonas reinhardtii.</a> The Plant Journal. 92 (2), 331-343 (2017).</li><li>Salem, M. A., Juppner, J., Bajdzienko, K., Giavalisco, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protocol:+a+fast,+comprehensive+and+reproducible+one-step+extraction+method+for+the+rapid+preparation+of+polar+and+semi-polar+metabolites,+lipids,+proteins,+starch+and+cell+wall+polymers+from+a+single+sample.">Protocol: a fast, comprehensive and reproducible one-step extraction method for the rapid preparation of polar and semi-polar metabolites, lipids, proteins, starch and cell wall polymers from a single sample.</a> Plant Methods. 12, 45 (2016).</li><li>Harris, E. H., Harris, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Chlamydomonas Sourcebook. 1, 248-249 (2009).</li><li>Roessner, U., Wagner, C., Kopka, J., Trethewey, R. N., Willmitzer, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Technical+advance:+simultaneous+analysis+of+metabolites+in+potato+tuber+by+gas+chromatography-mass+spectrometry.">Technical advance: simultaneous analysis of metabolites in potato tuber by gas chromatography-mass spectrometry.</a> The Plant Journal. 23 (1), 131-142 (2000).</li><li>Salem, M., Bernach, M., Bajdzienko, K., Giavalisco, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Simple+Fractionated+Extraction+Method+for+the+Comprehensive+Analysis+of+Metabolites,+Lipids,+and+Proteins+from+a+Single+Sample.">A Simple Fractionated Extraction Method for the Comprehensive Analysis of Metabolites, Lipids, and Proteins from a Single Sample.</a> Journal of Visualized Experiments. (124), (2017).</li><li>Bar-Nun, S., Ohad, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=[34]+Chloroplast+membrane+polypeptides.">[34] Chloroplast membrane polypeptides.</a> Methods in Enzymology. 69, 363-374 (1980).</li><li>Giavalisco, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteome+analysis+of+Arabidopsis+thaliana+by+two-dimensional+gel+electrophoresis+and+matrix-assisted+laser+desorption/ionisation-time+of+flight+mass+spectrometry.">Proteome analysis of Arabidopsis thaliana by two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionisation-time of flight mass spectrometry.</a> Proteomics. 5 (7), 1902-1913 (2005).</li><li>Bradford, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+and+sensitive+method+for+the+quantitation+of+microgram+quantities+of+protein+utilizing+the+principle+of+protein-dye+binding.">A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.</a> Analytical Biochemistry. 72, 248-254 (1976).</li><li>Rappsilber, J., Mann, M., Ishihama, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protocol+for+micro-purification,+enrichment,+pre-fractionation+and+storage+of+peptides+for+proteomics+using+StageTips.">Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips.</a> Nature Protocols. 2 (8), 1896-1906 (2007).</li><li>Bittremieux, W., Valkenborg, D., Martens, L., Laukens, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Computational+quality+control+tools+for+mass+spectrometry+proteomics.">Computational quality control tools for mass spectrometry proteomics.</a> Proteomics. 17 (3-4), 1600159 (2017).</li><li>Supek, F., Bosnjak, M., Skunca, N., Smuc, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=REVIGO+summarizes+and+visualizes+long+lists+of+gene+ontology+terms.">REVIGO summarizes and visualizes long lists of gene ontology terms.</a> PloS one. 6 (7), e21800 (2011).</li><li>Hauck, O. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uric+acid+accumulation+in+an+Arabidopsis+urate+oxidase+mutant+impairs+seedling+establishment+by+blocking+peroxisome+maintenance.">Uric acid accumulation in an Arabidopsis urate oxidase mutant impairs seedling establishment by blocking peroxisome maintenance.</a> Plant Cell. 26 (7), 3090-3100 (2014).</li><li>Caldana, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systemic+analysis+of+inducible+target+of+rapamycin+mutants+reveal+a+general+metabolic+switch+controlling+growth+in+Arabidopsis+thaliana.">Systemic analysis of inducible target of rapamycin mutants reveal a general metabolic switch controlling growth in Arabidopsis thaliana.</a> The Plant Journal. 73 (6), 897-909 (2013).</li><li>Avin-Wittenberg, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+analysis+of+the+role+of+autophagy+in+cellular+metabolism+and+energy+homeostasis+in+Arabidopsis+seedlings+under+carbon+starvation.">Global analysis of the role of autophagy in cellular metabolism and energy homeostasis in Arabidopsis seedlings under carbon starvation.</a> Plant Cell. 27 (2), 306-322 (2015).</li><li>Hummel, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultra+performance+liquid+chromatography+and+high+resolution+mass+spectrometry+for+the+analysis+of+plant+lipids.">Ultra performance liquid chromatography and high resolution mass spectrometry for the analysis of plant lipids.</a> Frontiers in Plant Science. 2, 54 (2011).</li><li>Giavalisco, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elemental+formula+annotation+of+polar+and+lipophilic+metabolites+using+(13)+C,+(15)+N+and+(34)+S+isotope+labelling,+in+combination+with+high-resolution+mass+spectrometry.">Elemental formula annotation of polar and lipophilic metabolites using (13) C, (15) N and (34) S isotope labelling, in combination with high-resolution mass spectrometry.</a> The Plant Journal. 68 (2), 364-376 (2011).</li><li>Watanabe, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+dissection+of+spatiotemporal+metabolic+shifts+in+primary,+secondary,+and+lipid+metabolism+during+developmental+senescence+in+Arabidopsis.">Comprehensive dissection of spatiotemporal metabolic shifts in primary, secondary, and lipid metabolism during developmental senescence in Arabidopsis.</a> Plant Physiology. 162 (3), 1290-1310 (2013).</li><li>Lambers, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteaceae+from+severely+phosphorus-impoverished+soils+extensively+replace+phospholipids+with+galactolipids+and+sulfolipids+during+leaf+development+to+achieve+a+high+photosynthetic+phosphorus-use-efficiency.">Proteaceae from severely phosphorus-impoverished soils extensively replace phospholipids with galactolipids and sulfolipids during leaf development to achieve a high photosynthetic phosphorus-use-efficiency.</a> New Phytologist. 196 (4), 1098-1108 (2012).</li><li>Degenkolbe, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+remodeling+of+the+lipidome+during+cold+acclimation+in+natural+accessions+of+Arabidopsis+thaliana.">Differential remodeling of the lipidome during cold acclimation in natural accessions of Arabidopsis thaliana.</a> The Plant Journal. 72 (6), 972-982 (2012).</li><li>Bromke, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liquid+chromatography+high-resolution+mass+spectrometry+for+fatty+acid+profiling.">Liquid chromatography high-resolution mass spectrometry for fatty acid profiling.</a> The Plant Journal. 81 (3), 529-536 (2015).</li><li>Li, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FAX1,+a+novel+membrane+protein+mediating+plastid+fatty+acid+export.">FAX1, a novel membrane protein mediating plastid fatty acid export.</a> PLOS Biology. 13 (2), e1002053 (2015).</li><li>Hemme, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systems-wide+analysis+of+acclimation+responses+to+long-term+heat+stress+and+recovery+in+the+photosynthetic+model+organism+Chlamydomonas+reinhardtii.">Systems-wide analysis of acclimation responses to long-term heat stress and recovery in the photosynthetic model organism Chlamydomonas reinhardtii.</a> Plant Cell. 26 (11), 4270-4297 (2014).</li><li>Sharma, D. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=UPLC-MS+analysis+of+Chlamydomonas+reinhardtii+and+Scenedesmus+obliquus+lipid+extracts+and+their+possible+metabolic+roles.">UPLC-MS analysis of Chlamydomonas reinhardtii and Scenedesmus obliquus lipid extracts and their possible metabolic roles.</a> Journal of Applied Phycology. 27 (3), 1149-1159 (2015).</li><li>Delgado, R., Munoz, Y., Pena-Cortes, H., Giavalisco, P., Bacigalupo, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diacylglycerol+activates+the+light-dependent+channel+TRP+in+the+photosensitive+microvilli+of+Drosophila+melanogaster+photoreceptors.">Diacylglycerol activates the light-dependent channel TRP in the photosensitive microvilli of Drosophila melanogaster photoreceptors.</a> The Journal of Neuroscience. 34 (19), 6679-6686 (2014).</li><li>Bozek, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organization+and+evolution+of+brain+lipidome+revealed+by+large-scale+analysis+of+human,+chimpanzee,+macaque,+and+mouse+tissues.">Organization and evolution of brain lipidome revealed by large-scale analysis of human, chimpanzee, macaque, and mouse tissues.</a> Neuron. 85 (4), 695-702 (2015).</li><li>Khrameeva, E. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neanderthal+ancestry+drives+evolution+of+lipid+catabolism+in+contemporary+Europeans.">Neanderthal ancestry drives evolution of lipid catabolism in contemporary Europeans.</a> Nature Communications. 5, 3584 (2014).</li><li>Bozek, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exceptional+evolutionary+divergence+of+human+muscle+and+brain+metabolomes+parallels+human+cognitive+and+physical+uniqueness.">Exceptional evolutionary divergence of human muscle and brain metabolomes parallels human cognitive and physical uniqueness.</a> PLOS Biology. 12 (5), e1001871 (2014).</li><li>Veyel, D., Erban, A., Fehrle, I., Kopka, J., Schroda, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rationales+and+approaches+for+studying+metabolism+in+eukaryotic+microalgae.">Rationales and approaches for studying metabolism in eukaryotic microalgae.</a> Metabolites. 4 (2), 184-217 (2014).</li><li>Allwood, J. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inter-laboratory+reproducibility+of+fast+gas+chromatography-electron+impact-time+of+flight+mass+spectrometry+(GC-EI-TOF/MS)+based+plant+metabolomics.">Inter-laboratory reproducibility of fast gas chromatography-electron impact-time of flight mass spectrometry (GC-EI-TOF/MS) based plant metabolomics.</a> Metabolomics. 5 (4), 479-496 (2009).</li><li>Feist, A. M., Herrgard, M. J., Thiele, I., Reed, J. L., Palsson, B. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstruction+of+biochemical+networks+in+microorganisms.">Reconstruction of biochemical networks in microorganisms.</a> Nature Reviews Microbiology. 7 (2), 129-143 (2009).</li></ol>

The authors have no disclosures.

In this article, we illustrated a robust and highly applicable extraction protocol for comprehensive lipidomics, metabolomics, starch and proteomics analysis from a single pellet of 10-15 x 106 cells. The method has been successfully implemented in several studies for a wide range of cells and tissues10,14,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36. Here, we presented a stepwise pipeline for multi-omics analysis of different biomolecules from a single sample harvested from Chlamydomonas reinhardtii (CC-1690 mt+) culture.
The protocol provides a robust and reproducible approach to process multiple samples at once, for the analysis of various biomolecules. However, a number of critical steps should be taken care off in order to minimize the technical variation. Firstly, harvesting of the cells should be done as swiftly as possible while maintaining the uniform conditions for all harvested samples to preserve the biological state of the cells. Though we used centrifugation to harvest the cells, alternate harvesting strategies can be used for harvesting of the samples. However, it is important to note that different harvesting strategies are known to influence the metabolic state of the cells37 hence, consistent harvesting approach must be used for all experimental samples. Secondly, it is important to avoid drying of the upper non-polar phase containing chlorophyll, since this can influence the levels of dissolved chlorophyll in the solvent affecting the normalization factor for the samples. Finally, care should be taken while removing the remaining polar phase to obtain the protein and starch pellet, to avoid disturbing the pellet which can influence the starch and protein content.
Thereby presented extraction protocol offers several benefits for multiple-omics data analysis. Besides minimizing the number of sample aliquots required, it also reduces the variation between the analytical results obtained for different biomolecules. This allows direct comparison of the results obtained from the primary metabolites, lipids and proteomic data. Similarly, the simultaneous extraction of multiple compound classes allows consistent and uniform normalization strategy of the different data sets. This is especially applicable if normalization is hard to achieve using dry or fresh weight38 or cell number.
The protocol can be implemented for routine screening of a complex biological sample. These holistic metabolomic, lipidomic and proteomic data sets can offer comprehensive information about systematic changes in the metabolism. Additionally, the data obtained from the proteomics analysis, provides insights into the quantitative (abundance) and qualitative (modifications) changes in proteins in relation to the metabolites. Hence, integrating omics data could reveal in-depth information about changes induced by genetic or biotic and/or abiotic perturbations of a biological system. Thus, elucidating molecular changes of specific metabolic pathways or cellular processes. Similarly, these high-throughput data can allow identification of targets for the metabolic engineering and refine or test predictions from genome-scale metabolic models37,39.

Microalgae are a rich source of natural products (e.g., fuels, human and animal nutrition, cosmetics and pharmacological substances). Numerous research efforts are carried out to enhance the efficiency of the production of high value products from microalgae1,2,3,4. Systems-level understanding of metabolism is a pre-requisite to improve the quality and the yield of natural products5,6,7. With the advent of functional genomic techniques and improved mass spectrometry methods, thousands of genes, transcripts, proteins, and metabolites can be monitored simultaneously. However, multiple samples are required for in-depth proteomics, lipidomics and metabolomics studies, which is often difficult to achieve in unicellular organisms, especially if time course studies are to be performed. Moreover, collection and processing of different sample aliquots in combination with different protocols to collect the highly complex omics data (i.e., proteomic, lipidomic, and metabolomics) introduces variability, thus making the integration of data a challenging task.
Chlamydomonas provides not only an excellent microbial system for the investigation of cellular processes, but also a convenient model to study the coordination of cell cycle and metabolism. Accordingly, a strong coordination of the transcripts expression with cell cycle has been shown using high resolution transcriptome profiling of synchronized culture of Chlamydomonas8. About 80% of the analyzed transcripts exhibited robust periodicity over a 24 h cell cycle8. Likewise, dry weight, proteins, chlorophyll, amino acids and fatty acids of two different strains of Chlamydomonas were shown to correlate with cell division in a study where sampling was performed every 4 h9. Recently, it was reported that the metabolite and lipid dynamics of the cell shift based on specific phases of the cell cycle10. The subtle changes in different biomolecules were possible to monitor using a robust methyl tert-butyl ether (MTBE): methanol: water-based extraction method, which offers an ideal starting point for comprehensive multi-omics analysis10,11.
The presented protocol guides through a reproducible and efficient strategy10, for the simultaneous extraction of lipids, metabolites, proteins and starch from a single sample aliquot, for the time resolved metabolomic and lipidomic study of synchronous growing Chlamydomonas cultures. In addition to illustrating the robust and reproducible metabolic and lipidomic data10, here, the quality of the proteomic samples obtained from the same pellet is also demonstrated.

<table>
	<tbody>
		<tr>
			<td>Reagents and standards</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1,2-diheptadecanoyl-sn-glycero-3-phosphocholine (17:0 PC)</td>
			<td>Avanti Polar Lipids</td>
			<td>850360P</td>
			<td>Internal standard for lipids</td>
		</tr>
		<tr>
			<td>13C Sorbitol</td>
			<td>Sigma Aldrich</td>
			<td>605514</td>
			<td>Internal standard for metabolites, ISOTEC&#174; Stable Isotopes</td>
		</tr>
		<tr>
			<td>Ampicillin</td>
			<td>Sigma Aldrich</td>
			<td>A9393-5G</td>
			<td>Internal standard for metabolites</td>
		</tr>
		<tr>
			<td>Corticosterone</td>
			<td>Sigma Aldrich</td>
			<td>27840-500MG</td>
			<td>Internal standard for metabolites, HPLC grade</td>
		</tr>
		<tr>
			<td>Methanol (MeOH)</td>
			<td>Biosolve Chemicals</td>
			<td>13684102</td>
			<td>ULC-MS grade</td>
		</tr>
		<tr>
			<td>Methyl tert-butyl ether (MTBE)</td>
			<td>Biosolve Chemicals</td>
			<td>13890602</td>
			<td>HPLC grade</td>
		</tr>
		<tr>
			<td>Trypsin/Lys-C mix</td>
			<td>Promega</td>
			<td>V5072</td>
			<td>Enzymatic digestion of proteins</td>
		</tr>
		<tr>
			<td>Water</td>
			<td>Biosolve Chemicals</td>
			<td>23214102</td>
			<td>ULC-MS grade</td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 ml Safe-lock microcentrifuge tubes</td>
			<td>Eppendorf</td>
			<td>30120086</td>
			<td>Used for fractions</td>
		</tr>
		<tr>
			<td>2 ml Safe-lock microcentrifuge tubes</td>
			<td>Eppendorf</td>
			<td>30120094</td>
			<td>Used for sample extarction</td>
		</tr>
		<tr>
			<td>Balance</td>
			<td>Sartorius Corporation</td>
			<td>14 557 572</td>
			<td></td>
		</tr>
		<tr>
			<td>Fermenter system</td>
			<td>Glasbl&#228;serei M&#252;ller, Berlin, Germany</td>
			<td></td>
			<td>custom made fermenter of 800ml capacity</td>
		</tr>
		<tr>
			<td>Q-exactive HF Orbitrap Mass Spectrometer</td>
			<td>Thermo Scientific</td>
			<td>IQLAAEGAAPFALGMBFZ</td>
			<td></td>
		</tr>
		<tr>
			<td>Refrigerated microcentrifuge</td>
			<td>Eppendorf, model 5427R</td>
			<td>22620701</td>
			<td></td>
		</tr>
		<tr>
			<td>Reversed Phase (RP) Bridged Ethyl Hybrid (BEH) C8 column (100 mm&#215;2.1 mm containing 1.7 &#956;m diameter particles)</td>
			<td>Waters, Machester, UK</td>
			<td>186002878</td>
			<td>Analysis of lipids</td>
		</tr>
		<tr>
			<td>RP Charged Surface Hybrid (CSH) column (Waters) with an inner diameter of 75 &#956;m and a particle size of 1.7 &#956;m</td>
			<td>Waters, Machester, UK</td>
			<td>186007477</td>
			<td>Analysis of proteins</td>
		</tr>
		<tr>
			<td>RP High Strength Silica (HSS) T3 column (100 mm&#215;2.1 mm containing 1.8 &#956;m diameter particles)</td>
			<td>Waters, Machester, UK</td>
			<td>186003539</td>
			<td>Analysis of metabolites</td>
		</tr>
		<tr>
			<td>Shaker</td>
			<td>Eppendorf Thermomixer 5436</td>
			<td>2050-100-05</td>
			<td></td>
		</tr>
		<tr>
			<td>Sonicator</td>
			<td>USC 300 TH</td>
			<td>142-0084</td>
			<td>standard preset sonication power at 45kHz</td>
		</tr>
		<tr>
			<td>UPLC system</td>
			<td>Waters Acquity UPLC system (Waters, Machester, UK)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum concentrator</td>
			<td>Scan Speed Maxi Vac Alpha Evaporators</td>
			<td>7.008.500.002</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex mixer</td>
			<td>Vortex-Genie 2, Model G560</td>
			<td>SI-0236</td>
			<td></td>
		</tr>
		<tr>
			<td>Z2 Coulter Particle Count and Size Analyzer</td>
			<td>Beckman Coulter</td>
			<td>6605700</td>
			<td>particle (cells) volume and number analyser</td>
		</tr>
	</tbody>
</table>

a multi-omics extraction method for the in-depth analysis of synchronized cultures of the green alga chlamydomonas reinhardtii

Microalgae have been the focus of research for their applications in the production of high value compounds, food and fuel. Moreover, they are valuable photosynthetic models facilitating the understanding of the basic cellular processes. System wide studies enable comprehensive and in-depth understanding of molecular functions of the organisms. However, multiple independent samples and protocols are required for proteomics, lipidomics and metabolomics studies introducing higher error and variability. A robust high throughput extraction method for the simultaneous extraction of chlorophyll, lipids, metabolites, proteins and starch from a single sample of the green alga Chlamydomonas reinhardtii is presented here. The illustrated experimental setup is for Chlamydomonas cultures synchronized using 12 h/12 h light/dark conditions. Samples were collected over a 24 h cell cycle to demonstrate that the metabolites, lipids and starch data obtained using various analytical platforms are well conformed. Furthermore, protein samples collected using the same extraction protocol were used to conduct detailed proteomics analysis to evaluate their quality and reproducibility. Based on the data, it can be inferred that the illustrated method provides a robust and reproducible approach to advance understanding of various biochemical pathways and their functions with greater confidence for both basic and applied research.

Chlamydomonas reinhardtii CC-1690 culture synchronization 
To demonstrate the representative results for the given protocol, we present the example multi-omics data obtained after harvesting and extraction of samples from synchronized Chlamydomonas reinhardtii cultures10. Synchronized cultures of Chlamydomonas comprise of cells belonging to uniform growth phase at a specific time point. The Chlamydomonas cultures were synchronized at 12 h/12 h light/dark cycle, 34 &#176;C with the light intensity of 200 &#181;mol&#183;m-2&#183;s-1 and the CO2 concentration of2%, v/v, described as optimal concentration for strain CC-1690 mt+10. These conditions had been previously optimized and validated using various cell cycle parameters10. Figure 1 displays cell size distribution measured with Coulter Counter at distinct time points of synchronized cultures. A shift in the cell volume can be observed as the cells grow in size throughout light phase, followed by the release of daughter cells starting at the end of light phase from 10 h. Once all the daughter cells are released, shift in the cell volume can be observed as the newly released daughter cells are disposed to begin the next cycle 10 (Figure 1).
Sample-harvesting, -handling and -extraction 
Rapid harvesting of samples is carried out using centrifugation and after discarding the supernatant, the pellets can be stored at -80 &#176;C until extraction. As described above (step 5), MTBE extraction results in three distinct phases: a) organic phase was used to measure lipids as well as chlorophyll levels (normalization factor), b) polar phase was collected to measure metabolites on GCMS while, c) the pellet was used to measure starch content and proteins. An overview of the distribution of different phases and their employment is illustrated in Figure 2.
Polar and non-polar metabolites 
Based on the GCMS analysis of the polar fraction, 65 metabolites were annotated, covering amino acids, nucleic acids, intermediates of glycolysis, gluconeogenesis, tricarboxylic acid cycle, pentose phosphate pathway and polyamines (Figure 3A). The LCMS analysis of neutral phase containing lipids led to the identification of 204 distinct lipid species covering various lipid classes namely phosphatidylglycerols, phosphatidylethanolamine, sulfoquinovosyl diacylglycerols, monogalactosyldiacylglycerols, digalactosyldiacylglycerols, diacylglyceryltrimethylhomoserine, fatty acids, diacylglycerides and triacylglycerides. To visualize the global shifts in the metabolites and lipids across cell cycle, principal component analysis (PCA) was used. The PCA displays a separation of light and dark phases for both metabolomics and lipidomic data. Moreover, a semi-cyclic (partially open circle) can be noticed for both data (Figure 3C,D). The partial gap in the circular pattern is attributed to the fact that the samples at 24 h of the cell cycle were collected under dark in contrast to the samples collected in the beginning of cell cycle after 0.25 h of exposure to the light (Figure 3C,D).
Protein and starch analysis 
To examine the quality of the protein pellet obtained as a result of MTBE extraction, 6 samples were used for proteomic analysis. The quality of the proteomics data obtained by digesting 50 &#181;g protein/sample, was examined using a computational quality control tool -Proteomics quality control (PTXQC)19, indicating reproducible and high quality of proteomics data obtained from all replicates (Supplementary Figure 1). The molecular functional coverage of proteins was examined using REVIGO20. An overview of functional enrichment of the 2463 identified proteins (see Table 2), is presented in Figure 4A. The remaining pellet after protein extraction was used for reproducible quantification of starch as indicated by low standard deviation among various replicates (Figure 4B).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59547/59547fig1.jpg" /> 
Figure 1: Illustrative example of the changes in the cell volume across different phases of cell cycle in Chlamydomonas reinhardtii. The x-axis representing the cell volume while y-axis representing the cell number. <a href="https://www.jove.com/files/ftp_upload/59547/59547fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59547/59547fig2.jpg" /> 
Figure 2: Illustrated workflow for the employment of different phases during multi-omics extraction cell pellets. The figure has been reused from Juppner, J.et al.10. <a href="https://www.jove.com/files/ftp_upload/59547/59547fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59547/59547fig3.jpg" /> 
Figure 3: Representative example of metabolites and lipids identified using the described protocol. (A) Metabolite classes identified by GCMS analysis. (B) Lipid species belonging to different classes identified by LCMS analysis. (C) Principle component analysis of the metabolite levels across 24 h cell cycle. (D) Principle component analysis of the lipids across 24 h cell cycle. <a href="https://www.jove.com/files/ftp_upload/59547/59547fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/59547/59547fig4.jpg" /> 
Figure 4: Representative example of the protein and starch data. A) Molecular functional enrichment of the proteins identified using LCMS analysis, treemap drawn using REVIGO 20 B) representative starch data displaying the reproducibility of the protocol. <a href="https://www.jove.com/files/ftp_upload/59547/59547fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Figure 1: Customized design of the fermenter system for the temperature and aeration controlled synchronous growth of Chlamydomonas cultures. The figure has been reused from Juppner, J.et al.10. <a href="https://www.jove.com/files/ftp_upload/59547/S1.eps">Please click here to download this file.</a>
Supplementary Figure 2: Representative outcome for proteomics data quality. Heatmap plotted using computational PTXQC tool19. <a href="https://www.jove.com/files/ftp_upload/59547/S2.eps">Please click here to download this file.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Time (min)</td>
			<td>% Buffer B to Buffer A</td>
			<td></td>
		</tr>
		<tr>
			<td>0 to 15 min</td>
			<td>Linear gradient from 0 to3%</td>
			<td>Buffer A: 0.1% formic acid in UPLC grade water</td>
		</tr>
		<tr>
			<td>15 to 75 min</td>
			<td>Linear gradient from 3% to 30%</td>
			<td>Buffer B: 0.1% formic acid in 60% UPLC grade acetonitrile</td>
		</tr>
		<tr>
			<td>75 to 90 min</td>
			<td>Linear gradient from 30% to 40%</td>
			<td>Flow rate 300 nL/min</td>
		</tr>
		<tr>
			<td>90 to 94 min</td>
			<td>Linear gradient from 40%&#160; to 90%</td>
			<td>Injection volume 4 &#181;L</td>
		</tr>
		<tr>
			<td>94 to104 min</td>
			<td>wash column with 90%</td>
			<td></td>
		</tr>
		<tr>
			<td>105 to 120 min</td>
			<td>Equilibrate the column for 15 min at 3%</td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 1: Liquid chromatography of peptide samples, gradient parameters.
Table 2: List of proteins identified after LCMS/MS analysis. <a href="https://www.jove.com/files/ftp_upload/59547/Table_2.xlsx">Please click here to download this file.</a>

Watch this Scientific Journal Video about A Multi-Omics Extraction Method for the In-Depth Analysis of Synchronized Cultures of the Green Alga Chlamydomonas reinhardtii at JoVE.com

A Multi-Omics Extraction Method for the In-Depth Analysis of Synchronized Cultures of the Green Alga Chlamydomonas reinhardtii

System-wide analysis of multiple biomolecules is crucial to gain functional and mechanistic insights into biological processes. Hereby, an extensive protocol is described for high throughput extraction of lipids, metabolites, proteins and starch from a single sample harvested from synchronized Chlamydomonas culture.

A Multi-Omics Extraction Method for the In-Depth Analysis of Synchronized Cultures of the Green Alga Chlamydomonas reinhardtii

Microalgae have been the focus of research for their applications in the production of high value compounds, food and fuel. Moreover, they are valuable ...

a-multi-omics-extraction-method-for-depth-analysis-synchronized

Research

JoVE Journal

Developmental Biology

7.5K Views.  Max Planck Institute of Molecular Plant Physiology. System-wide analysis of multiple biomolecules is crucial to gain functional and mechanistic insights into biological processes. Hereby, an extensive protocol is described for high throughput extraction of lipids, metabolites, proteins and starch from a single sample harvested from synchronized Chlamydomonas culture.

Video: A Multi-Omics Extraction Method for the In-Depth Analysis of Synchronized Cultures of the Green Alga Chlamydomonas reinhardtii

Application of the DNA-Specific Stain Methyl Green in the Fluorescent Labeling of Embryos

Methyl green has long been known as a histological stain with a specific affinity for DNA, although its fluorescent properties have remained unexplored until recently. In this article, we illustrate the method for preparing a methyl green aqueous stock solution, that when diluted can be used as a very convenient fluorescent nuclear label for fixed cells and tissues. Easy procedures to label whole zebrafish and chick embryos are detailed, and examples of images obtained shown. Methyl green is maximally excited by red light, at 633 nm, and emits with a relatively sharp spectrum that peaks at 677 nm. It is very inexpensive, non-toxic, highly stable in solution and very resistant to photobleaching when bound to DNA. Its red emission allows for unaltered high resolution scanning confocal imaging of nuclei in thick specimens. Finally, this methyl green staining protocol is compatible with other cell staining procedures, such as antibody labeling, or actin filaments labeling with fluorophore-conjugated phalloidin.

A method for fluorescent staining of fixed biological material with the specific DNA label methyl green is described. Methyl green is used in a diluted aqueous solution and is very resistant to photobleaching. Its far-red emission allows for deep specimen imaging, making it particularly adequate for whole embryos.

Methyl green has long been known as a histological stain with a specific affinity for DNA, although its fluorescent properties have remained unexplored ...

Using Confocal Analysis of Xenopus laevis to Investigate Modulators of Wnt and Shh Morphogen Gradients

This protocol describes a method to visualise ligands distributed across a field of cells. The ease of expressing exogenous proteins, together with the large size of their cells in early embryos, make Xenopus laevis a useful model for visualising GFP-tagged ligands. Synthetic mRNAs are efficiently translated after injection into early stage Xenopus embryos, and injections can be targeted to a single cell. When combined with a lineage tracer such as membrane tethered RFP, the injected cell (and its descendants) that are producing the overexpressed protein can easily be followed. This protocol describes a method for the production of fluorescently tagged Wnt and Shh ligands from injected mRNA. The methods involve the micro dissection of ectodermal explants (animal caps) and the analysis of ligand diffusion in multiple samples. By using confocal imaging, information about ligand secretion and diffusion over a field of cells can be obtained. Statistical analyses of confocal images provide quantitative data on the shape of ligand gradients. These methods may be useful to researchers who want to test the effects of factors that may regulate the shape of morphogen gradients.

Using Confocal Analysis of Xenopus laevis to Investigate Modulators of Wnt and Shh Morphogen Gradients

The manuscript here provides a simple set of methods for analysing the secretion and diffusion of fluorescently tagged ligands in Xenopus. This provides a context for testing the ability of other proteins to modify ligand distribution and allowing experiments that may give insight into mechanisms regulating morphogen gradients.

This protocol describes a method to visualise ligands distributed across a field of cells. The ease of expressing exogenous proteins, together with the ...

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

Differentiation of the SH-SY5Y Human Neuroblastoma Cell Line

Having appropriate in vivo and in vitro systems that provide translational models for human disease is an integral aspect of research in neurobiology and the neurosciences. Traditional in vitro experimental models used in neurobiology include primary neuronal cultures from rats and mice, neuroblastoma cell lines including rat B35 and mouse Neuro-2A cells, rat PC12 cells, and short-term slice cultures. While many researchers rely on these models, they lack a human component and observed experimental effects could be exclusive to the respective species and may not occur identically in humans. Additionally, although these cells are neurons, they may have unstable karyotypes, making their use problematic for studies of gene expression and reproducible studies of cell signaling. It is therefore important to develop more consistent models of human neurological disease. 
The following procedure describes an easy-to-follow, reproducible method to obtain homogenous and viable human neuronal cultures, by differentiating the chromosomally stable human neuroblastoma cell line, SH-SY5Y. This method integrates several previously described methods1-4 and is based on sequential removal of serum from media. The timeline includes gradual serum-starvation, with introduction of extracellular matrix proteins and neurotrophic factors. This allows neurons to differentiate, while epithelial cells are selected against, resulting in a homogeneous neuronal culture. Representative results demonstrate the successful differentiation of SH-SY5Y neuroblastoma cells from an initial epithelial-like cell phenotype into a more expansive and branched neuronal phenotype. This protocol offers a reliable way to generate homogeneous populations of neuronal cultures that can be used for subsequent biochemical and molecular analyses, which provides researchers with a more accurate translational model of human infection and disease.

It is critical in neurobiology and neurovirology to have a reliable, replicable in vitro system that serves as a translational model for what occurs in vivo in human neurons. This protocol describes how to culture and differentiate SH-SY5Y human neuroblastoma cells into viable neurons for use in in vitro applications.

Having appropriate in vivo and in vitro systems that provide translational models for human disease is an integral aspect of research in neurobiology and ...

A Method for Lineage Tracing of Corneal Cells Using Multi-color Fluorescent Reporter Mice

Lineage tracing experiments define the origin, fate and behavior of cells in a specific tissue or organism. This technique has been successfully applied for many decades, revealing seminal findings in developmental biology. More recently, it was adopted by stem cell biologists to identify and track different stem cell populations with minimal experimental intervention. The recent developments in mouse genetics, the availability of a large number of mouse strains, and the advancements in fluorescent microscopy allow the straightforward design of powerful lineage tracing systems for various tissues with basic expertise, using commercially available tools. We have recently taken advantage of this powerful methodology to explore the origin and fate of stem cells at the ocular surface using R26R-Confetti mouse. This model offers a multi-color genetic system, for the expression of 4 fluorescent genes in a random manner. Here we describe the principles of this methodology and provide an adaptable protocol for designing lineage tracing experiments; specifically for the corneal epithelium as well as for other tissues.

In this article we describe the principles for designing and performing a multi-color lineage tracing experiment using R26R-Confetti mice. We provide a specific protocol for tracking corneal epithelial cells which can be modified for other tissues of interest.

Lineage tracing experiments define the origin, fate and behavior of cells in a specific tissue or organism. This technique has been successfully applied ...

Culture of Murine Embryonic Metatarsals: A Physiological Model of Endochondral Ossification

The fundamental process of endochondral ossification is under tight regulation in the healthy individual so as to prevent disturbed development and/or longitudinal bone growth. As such, it is imperative that we further our understanding of the underpinning molecular mechanisms involved in such disorders so as to provide advances towards human and animal patient benefit. The mouse metatarsal organ explant culture is a highly physiological ex vivo model for studying endochondral ossification and bone growth as the growth rate of the bones in culture mimic that observed in vivo. Uniquely, the metatarsal organ culture allows the examination of chondrocytes in different phases of chondrogenesis and maintains cell-cell and cell-matrix interactions, therefore providing conditions closer to the in vivo situation than cells in monolayer or 3D culture. This protocol describes in detail the intricate dissection of embryonic metatarsals from the hind limb of E15 murine embryos and the subsequent analyses that can be performed in order to examine endochondral ossification and longitudinal bone growth.

We present a protocol to dissect and culture embryonic day 15 (E15) murine metatarsal bones. This highly physiological ex vivo model of endochondral ossification provides conditions closer to the in vivo situation than cells in monolayer or 3D culture and is a vital tool for investigating bone growth and development.

The fundamental process of endochondral ossification is under tight regulation in the healthy individual so as to prevent disturbed development and/or ...

Quantifying Branching Density in Rat Mammary Gland Whole-mounts Using the Sholl Analysis Method

An increasing number of studies are utilizing the rodent mammary gland as an endpoint for assessing the developmental toxicity of a chemical exposure. The effects these exposures have on mammary gland development are typically evaluated using either basic dimensional measurements or by scoring morphological characteristics. However, the broad range of methods for interpreting developmental changes could lead to inconsistent translations across laboratories. A common method of assessment is needed so that proper interpretations can be formed from data being compared across studies. The present study describes the application of the Sholl analysis method to quantify mammary gland branching characteristics. The Sholl method was originally developed for use in quantifying neuronal dendritic patterns. By using ImageJ, an open-source image analysis software package, and a plugin developed for this analysis, the mammary gland branching density and the complexity of a mammary gland from a peripubertal female rat were determined. The methods described here will enable the use of the Sholl analysis as an effective tool for quantifying an important characteristic of mammary gland development.

Mammary gland development in the rodent has typically been evaluated using descriptive assessments or by measuring basic physical attributes. Branching density is an indicator of mammary development that is difficult to quantify objectively. This protocol describes a reliable method for the quantitative assessment of mammary gland branching characteristics.

An increasing number of studies are utilizing the rodent mammary gland as an endpoint for assessing the developmental toxicity of a chemical exposure. The ...

A Seminiferous Tubule Squash Technique for the Cytological Analysis of Spermatogenesis Using the Mouse Model

Meiotic progression in males is a process that requires the concerted action of a number of highly regulated cellular events. Errors occurring during meiosis can lead to infertility, pregnancy loss or genetic defects. Commencing at the onset of puberty and continuing throughout adulthood, continuous semi-synchronous waves of spermatocytes undergo spermatogenesis and ultimately form haploid sperm. The first wave of mouse spermatocytes undergoing meiotic initiation appear at day 10 post-partum (10 dpp) and are released into the lumen of seminiferous tubules as mature sperm at 35 dpp. Therefore, it is advantageous to utilize mice within this developmental time-window in order to obtain highly enriched populations of interest. Analysis of rare cell stages is more difficult in older mice due to the contribution of successive spermatogenic waves, which increase the diversity of the cellular populations within the tubules. The method described here is an easily implemented technique for the cytological evaluation of the cells found within the seminiferous tubules of mice, including spermatogonia, spermatocytes, and spermatids. The tubule squash technique maintains the integrity of isolated male germ cells and allows examination of cellular structures that are not easily visualized with other techniques. To demonstrate the possible applications of this tubule squash technique, spindle assembly was monitored in spermatocytes progressing through the prophase to metaphase I transition (G2/MI transition). In addition, centrosome duplication, meiotic sex chromosome inactivation (MSCI), and chromosome bouquet formation were assessed as examples of the cytological structures that can be observed using this tubule squash method. This technique can be used to pinpoint specific defects during spermatogenesis that are caused by mutation or exogenous perturbation, and thus, contributes to our molecular understanding of spermatogenesis.

The goal of this tubule squash technique is to rapidly assess cytological features of developing mouse spermatocytes while preserving cellular integrity. This method allows for the study of all stages of spermatogenesis, and can be easily implemented alongside other biochemical and molecular biological approaches for the study of mouse meiosis.

Meiotic progression in males is a process that requires the concerted action of a number of highly regulated cellular events. Errors occurring during ...

Three-dimensional Rendering and Analysis of Immunolabeled, Clarified Human Placental Villous Vascular Networks

Nutrient and gas exchange between mother and fetus occurs at the interface of the maternal intervillous blood and the vast villous capillary network that makes up much of the parenchyma of the human placenta. The distal villous capillary network is the terminus of the fetal blood supply after several generations of branching of vessels extending out from the umbilical cord. This network has a contiguous cellular sheath, the syncytial trophoblast barrier layer, which prevents mixing of fetal blood and the maternal blood in which it is continuously bathed. Insults to the integrity of the placental capillary network, occurring in disorders such as maternal diabetes, hypertension and obesity, have consequences that present serious health risks for the fetus, infant, and adult. To better define the structural effects of these insults, a protocol was developed for this study that captures capillary network structure on the order of 1 - 2 mm3 wherein one can investigate its topological features in its full complexity. To accomplish this, clusters of terminal villi from placenta are dissected, and the trophoblast layer and the capillary endothelia are immunolabeled. These samples are then clarified with a new tissue clearing process which makes it possible to acquire confocal image stacks to z- depths of ~1 mm. The three-dimensional renderings of these stacks are then processed and analyzed to generate basic capillary network measures such as volume, number of capillary branches, and capillary branch end points, as validation of the suitability of this approach for capillary network characterization.

This study presents a protocol for the reversible tissue clearing, immunostaining, 3D-rendering and analysis of vascular networks in human placenta villi samples on the order of 1 - 2 mm3.

Nutrient and gas exchange between mother and fetus occurs at the interface of the maternal intervillous blood and the vast villous capillary network that ...

Three-dimensional Organotypic Cultures of Vestibular and Auditory Sensory Organs

The sensory organs of the inner ear are challenging to study in mammals due to their inaccessibility to experimental manipulation and optical observation. Moreover, although existing culture techniques allow biochemical perturbations, these methods do not provide a means to study the effects of mechanical force and tissue stiffness during development of the inner ear sensory organs. Here we describe a method for three-dimensional organotypic culture of the intact murine utricle and cochlea that overcomes these limitations. The technique for adjustment of a three-dimensional matrix stiffness described here permits manipulation of the elastic force opposing tissue growth. This method can therefore be used to study the role of mechanical forces during inner ear development. Additionally, the cultures permit virus-mediated gene delivery, which can be used for gain- and loss-of-function experiments. This culture method preserves innate hair cells and supporting cells and serves as a potentially superior alternative to the traditional two-dimensional culture of vestibular and auditory sensory organs.

Three-dimensional organotypic cultures of the murine utricle and cochlea in optically clear collagen I gels preserve innate tissue morphology, allow for mechanical stimulation through adjustment of matrix stiffness, and permit virus-mediated gene delivery.

The sensory organs of the inner ear are challenging to study in mammals due to their inaccessibility to experimental manipulation and optical observation. ...