This research was funded by CSIC I+D 2020 grant No. 282, FVF 2017 grant No. 210, and PEDECIBA (Mar&#237;a Martha Sainz).

1. Plant growth and rhizobia inoculation
<ol>
	<li>To generate the required nodules, sow the soybean seeds of choice in the selected substrate in a growth chamber under controlled conditions. 
	NOTE: In this protocol, seeds were sown in a 0.5 L plastic bottle filled with a mix of sand:vermiculite (1:1). The growth chamber was set with a day/night cycle temperature of 28 &#176;C /20 &#176;C, respectively, and a light/darkness photoperiod of 16 h/8 h, respectively. The photosynthetically active radiation intensity was 620 &#181;mol&#183;m&#8722;2&#183;s&#8722;1.</li>
	<li>Prepare in advance liquid Yeast-Extract Mannitol (YEM)-medium (see the Table of Materials). Autoclave the medium.</li>
	<li>On the same day of seed sowing, inoculate one flask containing 100 mL of YEM with the Bradyrhizobium elkanii U1302 strain. Incubate the flask at 28 &#176;C on an orbital shaker at 100 rpm.
	<ol>
		<li>Let the rhizobia grow for 2 days and inoculate the seedlings with 2 mL of the culture.</li>
	</ol>
	</li>
</ol>
2. Water deficit treatment (optional)
NOTE: This protocol outlines the water deficit treatment of the soybean plants. This part can be changed or omitted entirely depending on the experimental question at hand.
<ol>
	<li>Grow the soybean seedlings for 19 days (V2-3 developmental stage) without water restriction. Keep the substrate at field capacity with B&#38;D-medium17 supplemented with 0.5 mM KNO3.</li>
	<li>On day 20, withdraw watering. 
	NOTE: Here, the water content was measured daily by gravimetry (water gravimetric content) during the growth and water deficit periods.</li>
	<li>Measure stomatal conductance in triplicate daily on the abaxial leaf surface with a porometer, as instructed by the manufacturer (see the Table of Materials), from day 20 until the end of the water deficit period. 
	&#8203;NOTE: The end of the water deficit period was determined individually for each plant when the stomatal conductance value was approximately 50% of the one obtained on day 20 from sowing.</li>
</ol>
3. Nodule harvest
<ol>
	<li>Collect liquid nitrogen in a clean container and label 15 mL tubes (one for each plant). 
	CAUTION: Handle liquid nitrogen wearing cryo gloves, face shields, and safety goggles.</li>
	<li>Retrieve each plant individually. Cut and discard the aerial part. Thoroughly wash the root with water to remove any remaining substrate.</li>
	<li>Detach each nodule from the root and collect them in a prechilled 15 mL tube. Snap-freeze the tubes and store them at &#8722;80 &#176;C.</li>
</ol>
4. Preparation of cytosolic extracts
NOTE: The final aim of this protocol is to obtain high-quality total RNA (TOTAL) and polysome-associated RNA (PAR). Therefore, work under conditions that prevent RNA degradation, always keeping the samples at 4 &#176;C and using RNase-free laboratory equipment and solutions. Unless specified, all the solutions are prepared with sterile ultrapure water.
<ol>
	<li>Preparation of buffer stock solutions
	<ol>
		<li>Prepare the autoclavable stock solutions listed in Table 1, autoclave them for 15 min, and keep at RT.</li>
		<li>Prepare the filter-sterilizable stock solutions listed in Table 1, filter-sterilize them, and keep them at &#8722;20 &#176;C.</li>
	</ol>
	</li>
	<li>Prepare polysome extraction buffer (PEB, see Table 1) and keep it on ice. 
	NOTE: The volumes given are for six samples.</li>
	<li>Cool the centrifuge to 4 &#176;C and place 2 mL microcentrifuge tubes on ice.</li>
	<li>Weigh approximately 0.2 g of intact nodules in a weighing dish and transfer them to a precooled 2 mL tube. 
	NOTE: Perform this step quickly to avoid the thawing of the samples. Alternatively, an estimated nodule volume of 0.4-0.5 mL can be used instead of the 0.2 g.</li>
	<li>Add 1.2 mL of PEB, let the sample thaw for 2 min, and homogenize with a tissue grinder until complete disruption and homogenization of the nodules. 
	NOTE: Be sure to grind the nodules once thawed as they will be soft enough to be easily disrupted with tissue grinders (see the Table of Materials) in microcentrifuge tubes. Frozen nodules are difficult to grind. Also, it is recommended to place the tubes in a benchtop cooler (0 &#176;C) for proper homogenization while keeping samples cool.</li>
	<li>Incubate the samples on ice with gentle agitation for 10 min or until all samples have been processed.</li>
	<li>Centrifuge at 16,000 &#215; g for 15 min at 4 &#176;C to pellet the debris. Recover the supernatant and repeat the centrifuge step.</li>
	<li>Carefully recover the clarified cytosolic extract and transfer a 200 &#181;L aliquot to a clean 1.5 mL microcentrifuge tube for TOTAL isolation (TOTAL samples; see section 7).</li>
</ol>
5. Preparation of sucrose cushions
NOTE: This protocol uses a two-layer sucrose cushion (12% and 33.5%) in 13.2 mL ultracentrifuge tubes (see the Table of Materials). All solutions are prepared with sterile ultrapure water.
<ol>
	<li>Prepare the sucrose and salt stock solutions.
	<ol>
		<li>Prepare 100 mL of 2 M sucrose solution (68.5%, Table 1).</li>
		<li>Prepare 10x salt solution (Table 1).</li>
	</ol>
	</li>
	<li>Prepare the two layers of the sucrose cushion as described in Table 2. See Table 1 for the composition of the antibiotic (CHX and CHL) stock solutions. 
	NOTE: The volumes given are for six cushions.</li>
	<li>Pour 4.5 mL of the 33.5% sucrose layer into the centrifuge tubes. Then, add 4.5 mL of the 12% layer carefully and slowly with a P1000 micropipette. Put the tubes on ice until the addition of the clarified cytosolic extract.</li>
</ol>
6. Polysome purification
<ol>
	<li>Load 1 mL of the clarified cytosolic extract (step 4.8) on top of the sucrose cushion by carefully pipetting onto the sidewall of the tube.</li>
	<li>Transfer the ultracentrifuge tubes to precooled buckets and centrifuge at 217,874 &#215; g&#160;(35,000 rpm in the referenced rotor [see the Table of Materials]) for 2 h at 4 &#176;C.</li>
	<li>Discard the remaining cytosolic extract and sucrose cushion, and resuspend the polysomal pellet with 200 &#181;L of precooled RB (previously prepared according to Table 1).</li>
	<li>Incubate for 30 min on ice and then transfer the polysomal resuspension to 1.5 mL precooled tubes. Proceed with the RNA extraction (PAR samples).</li>
</ol>
7. RNA extraction and quality control
NOTE: This step is performed for TOTAL (step 4.8) and PAR samples (step 6.4).
<ol>
	<li>Prepare 75% EtOH with sterile ultrapure water and cool it to &#8722;20 &#176;C. Additionally, cool chloroform and isopropanol to &#8722;20 &#176;C.</li>
	<li>Homogenize the samples with 750 &#181;L of the RNA isolation reagent and incubate for 5 min at RT.</li>
	<li>Add 200 &#181;L of cold chloroform and shake the tubes vigorously for 15 s. Incubate at RT for 10 min.</li>
	<li>Centrifuge at 12,000 &#215; g for 15 min at 4 &#176;C for phase separation. Transfer 500 &#181;L of the upper aqueous phase to a clean tube without disturbing the pink organic phase, and add 375 &#181;L of cold isopropanol and 0.5 &#181;L of RNase-free glycogen (see the Table of Materials). Mix thoroughly by pipetting up and down. 
	NOTE: Glycogen is a carrier used as a co-precipitant to increase nucleic acid recovery from alcohol precipitation.</li>
	<li>Incubate the mix for 10 min at 4 &#176;C and centrifuge at 12,000 &#215; g for 15 min. Discard the supernatant.</li>
	<li>Wash the RNA precipitate with 1 mL of cold 75% EtOH. Mix by brief vortexing. 
	NOTE: At this point, the samples can be stored at &#8722;20 &#176;C overnight.</li>
	<li>Centrifuge at 7,500 &#215; g for 5 min at 4 &#176;C, carefully remove the supernatant with a micropipette, and air-dry the RNA pellet.</li>
	<li>Dissolve the pellet in 50 &#181;L of RNase-free water and incubate at 65 &#176;C for 5 min.</li>
	<li>Assess the RNA concentration and integrity with highly sensitive capillary electrophoresis18 (see the Table of Materials) and/or electrophoresis on a 2% RNase-free agarose gel19 (see the Table of Materials). 
	NOTE: If the RNA samples are not going to be used immediately or are going to be sent to the sequencing facility for RNA-seq library preparation, it is recommended to use EtOH to precipitate them.</li>
</ol>
8. RNA precipitation
<ol>
	<li>Cool the centrifuge and EtOH to 4 &#176;C.</li>
	<li>Prepare 3 M sodium acetate.</li>
	<li>Prepare 70% EtOH with sterile ultrapure water and cool it to &#8722;20 &#176;C.</li>
	<li>Estimate the sample volume. Add 0.1 volumes of 3 M sodium acetate, 3 volumes of cold EtOH, and 0.5 &#181;L of RNase-free glycogen. Mix thoroughly.</li>
	<li>Leave at &#8722;20 &#176;C until needed. 
	&#8203;NOTE: RNA precipitated samples can be stored for up to 1 year at &#8722;80 &#176;C.</li>
</ol>
9. Standard pipeline for gene expression analysis
<ol>
	<li>Perform an average of 40M Illumina paired-end reads, with read length &#62;100 bp, for confident transcriptome and translatome data analysis. 
	NOTE: Standard RNA-seq data analysis includes the following steps 9.2-9.5.</li>
	<li>Carry out read quality inspection using FastQC (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/)&#160;or the multisample MultiQC20.</li>
	<li>Remove adaptor and low-quality sequences using either Trimmomatic21, BBDuk (http://jgi.doe.gov/data-and-tools/bb-tools/), cutadapt22, sickle23, or Trim Galore (https://www.bioinformatics.babraham.ac.uk/projects/trim_galore/), among others.</li>
	<li>If a reference genome is available for the species, carry out read mapping against the reference, followed by abundance quantification, using Bowtie224, TopHat225 or Salmon26, and featureCounts27, besides other open source tools.</li>
	<li>Perform statistical analysis for differential expressed genes identification using edgeR28, DESeq229, or limma30, among others. 
	NOTE: These open-source software can be used locally, via the command line. Alternatively, they can be operated through a web browser on a public server, such as Galaxy31 or GEOexplorer32, which offer a graphical user interface, so that no command line knowledge is required for their use.</li>
</ol>

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W., Bailey-Serres, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunopurification+of+polyribosomal+complexes+of+Arabidopsis+for+global+analysis+of+gene+expression.">Immunopurification of polyribosomal complexes of Arabidopsis for global analysis of gene expression.</a> Plant physiology. 138 (2), 624-635 (2005).</li><li>Mustroph, 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=Profiling+translatomes+of+discrete+cell+populations+resolves+altered+cellular+priorities+during+hypoxia+in+Arabidopsis.">Profiling translatomes of discrete cell populations resolves altered cellular priorities during hypoxia in Arabidopsis.</a> Proceedings of the National Academy of Sciences of the United States of America. 106 (44), 18843-11848 (2009).</li><li>Ingolia, N. T., Ghaemmaghami, S., Newman, J. R. S., Weissman, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+analysis+in+vivo+of+translation+with+nucleotide+resolution+using+ribosome+profiling.">Genome-wide analysis in vivo of translation with nucleotide resolution using ribosome profiling.</a> Science. 324 (5924), 218-223 (2009).</li><li>Brar, G. A., Weissman, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ribosome+profiling+reveals+the+what,+when,+where+and+how+of+protein+synthesis.">Ribosome profiling reveals the what, when, where and how of protein synthesis.</a> Nature Reviews. Molecular Cell Biology. 16 (11), 651-664 (2015).</li><li>Eastman, G., Smircich, P., Sotelo-Silveira, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Following+ribosome+footprints+to+understand+translation+at+a+genome+wide+level.">Following ribosome footprints to understand translation at a genome wide level.</a> Computational and Structural Biotechnology Journal. 16, 167-176 (2018).</li><li>Westermann, A. J., Gorski, S. A., Vogel, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual+RNA-seq+of+pathogen+and+host.">Dual RNA-seq of pathogen and host.</a> Nature Reviews Microbiology. 10 (9), 618-630 (2012).</li></ol>

The authors have no conflicts of interest.

Studying gene expression regulation at the translational level is critical to better comprehend different biological processes since the endpoint of cell gene expression is protein abundance13,14. This can be assessed by analyzing the translatome of the tissue or organism of interest for which the polysomal fraction should be purified and its associated mRNAs analyzed3,4,34</s...

Leguminous plants, such as soybean (Glycine max), can establish symbiosis with specific soil bacteria called rhizobia. This mutualistic relationship elicits the formation of novel organs, the symbiotic nodules, on the plant roots. The nodules are the plant organs hosting the bacteria and consist of host cells whose cytoplasm is colonized with a specialized form of rhizobia called bacteroids. These bacteroids catalyze the reduction of atmospheric nitrogen (N2) into ammonia, which is transferred to the plant in return for carbohydrates1,2.
Although this nitrogen-fixing symbiosis is one of the most well-studied plant-microbe symbioses, many aspects remain to be better understood, such as how plants subjected to different abiotic stress conditions modulate their interaction with their symbiotic partner and how this affects nodule metabolism. These processes could be better understood by analyzing the nodule translatome (i.e., the subset of messenger RNAs [mRNAs] actively translated). Polyribosomes or polysomes are complexes of multiple ribosomes associated with mRNA, commonly used to study translation3. The polysome profiling method consists of the analysis of the mRNAs associated with polysomes and has been successfully used to study the posttranscriptional mechanisms controlling gene expression that occurs in diverse biological processes4,5.
Historically, genome expression analysis has focused primarily on determining mRNA abundance6,7,8,9. However, there is a lack of correlation between transcript and protein levels due to the different stages of posttranscriptional regulation of gene expression, particularly translation10,11,12. Moreover, no dependence has been observed between the changes at the level of the transcriptome and those that occur at the level of the translatome13. The direct analysis of the set of mRNAs that are being translated allows a more accurate and complete measurement of the cell gene expression (whose endpoint is protein abundance) than the one obtained when only mRNA levels are analyzed14,15,16.
This protocol describes how plant-derived polysomes are purified from intact soybean nodules by differential centrifugation through a two-layer sucrose cushion (Figure 1). However, since bacteroid-derived ribosomes are also present in the nodules, a mix of ribosomes and RNA species are purified, even though the eukaryotic ones represent the main fraction (90%-95%). The subsequent RNA isolation, quantification, and quality control are also described (Figure 1). This protocol, in combination with RNA-seq, should provide experimental results on the translational regulation of eukaryotic mRNAs in a complex tissue such as the symbiotic nodule.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64269/64269fig01.jpg" /> 
Figure 1: Schematic overview of the proposed methodology for eukaryotic polysome purification from symbiotic nodules. The scheme gives an overview of the steps followed in the protocol from (1) plant growth and (2) nodule harvest to (3) preparation of the cytosolic extracts, (3) obtaining TOTAL samples and (4) PAR samples, and (5) RNA extraction and quality control. Abbreviations: PEB = polysome extraction buffer; RB = resuspension buffer; TOTAL = total RNA; PAR = polysome-associated mRNA. <a href="https://www.jove.com/files/ftp_upload/64269/64269fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Plant growth and rhizobia inoculation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Orbital shaker</td>
			<td>Daihan Scientific</td>
			<td></td>
			<td>Model SHO-1D</td>
		</tr>
		<tr>
			<td>YEM-medium</td>
			<td>Amresco</td>
			<td>J850 (yeast extract) 0122 (mannitol)</td>
			<td></td>
		</tr>
		<tr>
			<td>Water deficit treatment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>KNO3</td>
			<td>Merck</td>
			<td>221295</td>
			<td></td>
		</tr>
		<tr>
			<td>Porometer</td>
			<td>Decagon Device</td>
			<td></td>
			<td>Model SC-1</td>
		</tr>
		<tr>
			<td>Scalpel</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Preparation of cytosolic extracts</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Brij L23</td>
			<td>Sigma-Aldrich</td>
			<td>P1254</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Sigma</td>
			<td></td>
			<td>Model 2K15</td>
		</tr>
		<tr>
			<td>Chloranphenicol</td>
			<td>Sigma-Aldrich</td>
			<td>C0378</td>
			<td></td>
		</tr>
		<tr>
			<td>Cycloheximide</td>
			<td>Sigma-Aldrich</td>
			<td>C7698</td>
			<td></td>
		</tr>
		<tr>
			<td>DOC</td>
			<td>Sigma-Aldrich</td>
			<td>30970</td>
			<td></td>
		</tr>
		<tr>
			<td>DTT</td>
			<td>Sigma-Aldrich</td>
			<td>D9779</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma-Aldrich</td>
			<td>E3889</td>
			<td></td>
		</tr>
		<tr>
			<td>Igepal CA 360</td>
			<td>Sigma-Aldrich</td>
			<td>I8896</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Merck</td>
			<td>1.04936</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma-Aldrich</td>
			<td>M8266</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic tissue grinder</td>
			<td>Fisher Scientific</td>
			<td>12649595</td>
			<td></td>
		</tr>
		<tr>
			<td>PMSF</td>
			<td>Sigma-Aldrich</td>
			<td>P7626</td>
			<td></td>
		</tr>
		<tr>
			<td>PTE</td>
			<td>Sigma-Aldrich</td>
			<td>P2393</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>Invitrogen</td>
			<td>15504-020</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Sigma-Aldrich</td>
			<td>P1379</td>
			<td></td>
		</tr>
		<tr>
			<td>Weighing dish&#160;</td>
			<td>Deltalab</td>
			<td>1911103</td>
			<td></td>
		</tr>
		<tr>
			<td>Preparation of sucrose cushions</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Invitrogen</td>
			<td>15503022</td>
			<td></td>
		</tr>
		<tr>
			<td>SW 40 Ti rotor</td>
			<td>Beckman-Coulter</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ultracentrifuge</td>
			<td>Beckman-Coulter</td>
			<td></td>
			<td>Optima L-100K</td>
		</tr>
		<tr>
			<td>Ultracentrifuge tubes</td>
			<td>Beckman-Coulter</td>
			<td>344059</td>
			<td>13.2 mL tubes</td>
		</tr>
		<tr>
			<td>RNA extraction and quality control</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Thermo scientific</td>
			<td>R0492</td>
			<td></td>
		</tr>
		<tr>
			<td>Bioanalyzer</td>
			<td>Agilent</td>
			<td></td>
			<td>Model 2100. Eukaryote total RNA nano assay</td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>DI</td>
			<td>41191</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Dorwil</td>
			<td>UN1170</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td>Mallinckrodt</td>
			<td>3032-06</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycogen</td>
			<td>Sigma</td>
			<td>10814-010</td>
			<td></td>
		</tr>
		<tr>
			<td>TRIzol LS</td>
			<td>Ambion</td>
			<td>102960028</td>
			<td></td>
		</tr>
		<tr>
			<td>Miscellaneous</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon tubes 15 mL</td>
			<td>Biologix</td>
			<td>10-0152</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter tips 10 &#181;L</td>
			<td>BioPointe Scientific</td>
			<td>321-4050</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter tips 1000 &#181;L</td>
			<td>BioPointe Scientific</td>
			<td>361-1050</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter tips 20 &#181;L</td>
			<td>BioPointe Scientific</td>
			<td>341-4050</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter tips 200 &#181;L</td>
			<td>Tarsons</td>
			<td>528104</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge tubes 1.5 mL</td>
			<td>Tarsons</td>
			<td>500010-N</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge tubes 2.0 mL</td>
			<td>Tarsons</td>
			<td>500020-N</td>
			<td></td>
		</tr>
		<tr>
			<td>Sequencing company</td>
			<td>Macrogen</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile 250 mL flask</td>
			<td>Marienfeld</td>
			<td>4110207</td>
			<td></td>
		</tr>
	</tbody>
</table>

polysome purification from soybean symbiotic nodules

The aim of this protocol is to provide a strategy for studying the eukaryotic translatome of the soybean (Glycine max) symbiotic nodule. This paper describes methods optimized to isolate plant-derived polyribosomes and their associated mRNAs to be analyzed using RNA-sequencing. First, cytoplasmic lysates are obtained through homogenization in polysome- and RNA-preserving conditions from whole, frozen soybean nodules. Then, lysates are cleared by low-speed centrifugation, and 15% of the supernatant is used for total RNA (TOTAL) isolation. The remaining cleared lysate is used to isolate polysomes by ultracentrifugation through a two-layer sucrose cushion (12% and 33.5%). Polysome-associated mRNA (PAR) is purified from polysomal pellets after resuspension. Both TOTAL and PAR are evaluated by highly sensitive capillary electrophoresis to meet the quality standards of sequencing libraries for RNA-seq. As an example of a downstream application, after sequencing, standard pipelines for gene expression analysis can be used to obtain differentially expressed genes at the transcriptome and translatome levels. In summary, this method, in combination with RNA-seq, allows the study of the translational regulation of eukaryotic mRNAs in a complex tissue such as the symbiotic nodule.

The quantity and quality assessment of the TOTAL and PAR fractions purified with the abovementioned procedure is key to determining its success, since for most downstream applications, such as RNA sequencing, high-quality samples are fundamental for library preparation and sequencing. Moreover, the integrity of the RNA molecules allows the capture of a snapshot of the gene expression profile at the moment of sample collection18. In this context, an RNA integrity number (RIN) is obtained when perfo...

Watch this Scientific Journal Video about Polysome Purification from Soybean Symbiotic Nodules at JoVE.com

Polysome Purification from Soybean Symbiotic Nodules

This protocol describes a method for eukaryotic polysome purification from intact soybean nodules. After sequencing, standard pipelines for gene expression analysis can be used to identify differentially expressed genes at the transcriptome and translatome levels.

The aim of this protocol is to provide a strategy for studying the eukaryotic translatome of the soybean (Glycine max) symbiotic nodule. This paper ...

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Developmental Biology

1.4K Views.  Universidad de la República. This protocol describes a method for eukaryotic polysome purification from intact soybean nodules. After sequencing, standard pipelines for gene expression analysis can be used to identify differentially expressed genes at the transcriptome and translatome levels.

Video: Polysome Purification from Soybean Symbiotic Nodules

Three-dimensional Quantification of Dendritic Spines from Pyramidal Neurons Derived from Human Induced Pluripotent Stem Cells

Dendritic spines are small protrusions that correspond to the post-synaptic compartments of excitatory synapses in the central nervous system. They are distributed along the dendrites. Their morphology is largely dependent on neuronal activity, and they are dynamic. Dendritic spines express glutamatergic receptors (AMPA and NMDA receptors) on their surface and at the levels of postsynaptic densities. Each spine allows the neuron to control its state and local activity independently. Spine morphologies have been extensively studied in glutamatergic pyramidal cells of the brain cortex, using both in vivo approaches and neuronal cultures obtained from rodent tissues. Neuropathological conditions can be associated to altered spine induction and maturation, as shown in rodent cultured neurons and one-dimensional quantitative analysis 1. The present study describes a protocol for the 3D quantitative analysis of spine morphologies using human cortical neurons derived from neural stem cells (late cortical progenitors). These cells were initially obtained from induced&#160;pluripotent stem cells. This protocol allows the analysis of spine morphologies at different culture periods, and with possible comparison between induced&#160;pluripotent stem cells obtained from control individuals with those obtained from patients with psychiatric diseases.

Dendritic spines of pyramidal neurons are the sites of most excitatory synapses in mammalian brain cortex. This method describes a 3D quantitative analysis of spine morphologies in human cortical pyramidal glutamatergic neurons derived from induced&#160;pluripotent stem cells.

Dendritic spines are small protrusions that correspond to the post-synaptic compartments of excitatory synapses in the central nervous system. They are ...

A Quick and Efficient Method for the Purification of Endoderm Cells Generated from Human Embryonic Stem Cells

The differentiation capabilities of pluripotent stem cells such as embryonic stem cells (ESCs) allow a potential therapeutic application for cell replacement therapies. Terminally differentiated cell types could be used for the treatment of various degenerative diseases. In vitro differentiation of these cells towards tissues of the lung, liver and pancreas requires as a first step the generation of definitive endodermal cells. This step is rate-limiting for further differentiation towards terminally matured cell types such as insulin-producing beta cells, hepatocytes or other endoderm-derived cell types. Cells that are committed towards the endoderm lineage highly express a multitude of transcription factors such as FOXA2, SOX17, HNF1B, members of the GATA family, and the surface receptor CXCR4. However, differentiation protocols are rarely 100% efficient. Here, we describe a method for the purification of a CXCR4+ cell population after differentiation into the DE by using magnetic microbeads. This purification additionally removes cells of unwanted lineages. The gentle purification method is quick and reliable and might be used to improve downstream applications and differentiations.

Here, we describe a method for the purification of differentiated human embryonic stem cells that are committed towards the definitive endoderm for the improvement of downstream applications and further differentiations.

The differentiation capabilities of pluripotent stem cells such as embryonic stem cells (ESCs) allow a potential therapeutic application for cell ...

Affinity Labeling Detection of Endogenous Receptors from Zebrafish Embryos

By combining the powers of Affinity Labeling and Immunoprecipitation (AFLIP), a technique for the detection of low abundance receptors in zebrafish embryos has been implemented. This technique takes advantage of the selectivity and sensitivity conferred by affinity labeling of a given receptor by its ligand with the specificity of the immunoprecipitation. We have used AFLIP to detect the type III TGF-&#946; receptor (TGFBR3), also know as betaglycan, during early zebrafish development. AFLIP was instrumental in validating the efficacy of a TGFBR3 morphant zebrafish phenotype. In the first step, embryo protein extracts are prepared and used to generate 125I-TGF-&#946;2-TGFBR3 complexes that are purified by immunoprecipitation. Later, these complexes are covalently cross-linked and revealed using SDS-PAGE separation and autoradiography detection. This technique requires the availability of a labeled ligand for, and a specific antibody against, the receptor to be detected, and shall be easily adapted to identify any growth factor or cytokine receptor that meets these requirements.

A novel technique for the detection of low abundance endogenous receptors present in zebrafish embryos is described. We have named it AFLIP because it consists of affinity labeling of the receptor by its ligand linked to immunoprecipitation.

By combining the powers of Affinity Labeling and Immunoprecipitation (AFLIP), a technique for the detection of low abundance receptors in zebrafish ...

Establishment of Proliferative Tetraploid Cells from Nontransformed Human Fibroblasts

Polyploid (mostly tetraploid) cells are often observed in preneoplastic lesions of human tissues and their chromosomal instability has been considered to be responsible for carcinogenesis in such tissues. Although proliferative polyploid cells are requisite for analyzing chromosomal instability of polyploid cells, creating such cells from nontransformed human cells is rather challenging. Induction of tetraploidy by chemical agents usually results in a mixture of diploid and tetraploid populations, and most studies employed fluorescence-activated cell sorting or cloning by limiting dilution to separate tetraploid from diploid cells. However, these procedures are time-consuming and laborious. The present report describes a relatively simple protocol to induce proliferative tetraploid cells from normal human fibroblasts with minimum contamination by diploid cells. Briefly, the protocol is comprised of the following steps: arresting cells in mitosis by demecolcine (DC), collecting mitotic cells after shaking off, incubating collected cells with DC for an additional 3 days, and incubating cells in drug-free medium (They resume proliferation as tetraploid cells within several days). Depending on cell type, the collection of mitotic cells by shaking off might be omitted. This protocol provides a simple and feasible method to establish proliferative tetraploid cells from normal human fibroblasts. Tetraploid cells established by this method could be a useful model for studying chromosome instability and the oncogenic potential of polyploid human cells.

Although proliferative polyploid cells are necessary to analyze chromosomal instability of polyploid cells, creating such cells from nontransformed human cells is not easy. The present report describes relatively simple procedures to establish proliferative tetraploid cells free of a diploid population from normal human fibroblasts.

Polyploid (mostly tetraploid) cells are often observed in preneoplastic lesions of human tissues and their chromosomal instability has been considered to ...

A Standardized Approach for Multispecies Purification of Mammalian Male Germ Cells by Mechanical Tissue Dissociation and Flow Cytometry

Fluorescence-activated cell sorting (FACS) has been one of the methods of choice to isolate enriched populations of mammalian testicular germ cells. Currently, it allows the discrimination of up to 9 murine germ cell populations with high yield and purity. This high-resolution in discrimination and purification is possible due to unique changes in chromatin structure and quantity throughout spermatogenesis. These patterns can be captured by flow cytometry of male germ cells stained with fluorescent DNA-binding dyes such as Hoechst-33342 (Hoechst). Herein is a detailed description of a recently developed protocol to isolate mammalian testicular germ cells. Briefly, single cell suspensions are generated from testicular tissue by mechanical dissociation, double stained with Hoechst and propidium iodide (PI) and processed by flow cytometry. A serial gating strategy, including the selection of live cells (PI negative) with different DNA content (Hoechst intensity), is used during FACS sorting to discriminate up to 5 germ cell types. These include, with corresponding average purities (determined by microscopy evaluation): spermatogonia (66%), primary (71%) and secondary (85%) spermatocytes, and spermatids (90%), further separated into round (93%) and elongating (87%) subpopulations. Execution of the entire workflow is straightforward, allows the isolation of 4 cell types simultaneously with the appropriate FACS machine, and can be performed in less than 2 h. As reduced processing time is crucial to preserve the physiology of ex vivo cells, this method is ideal for downstream high-throughput studies of male germ cell biology. Moreover, a standardized protocol for multispecies purification of mammalian germ cells eliminates methodological sources of variables and allows a single set of reagents to be used for different animal models.

This work describes the standardization of a method to obtain purified germ cell populations from testicular tissue of different mammalian species. It is a straightforward protocol that combines mechanical testis dissociation, staining with Hoechst-33342 and propidium iodide, and FACS sorting, with wide applications in comparative studies of male reproductive biology.

Fluorescence-activated cell sorting (FACS) has been one of the methods of choice to isolate enriched populations of mammalian testicular germ cells. ...

Derivation of Stem Cell Lines from Mouse Preimplantation Embryos

Mouse embryonic stem cell (mESC) derivation is the process by which pluripotent cell lines are established from preimplantation embryos. These lines retain the ability to either self-renew or differentiate under specific conditions. Due to these properties, mESC are a useful tool in regenerative medicine, disease modeling, and tissue engineering studies. This article describes a simple protocol to obtain mESC lines with high derivation efficiencies (60-80%) by culturing blastocysts from permissive mouse strains on feeder cells in defined medium supplemented with leukemia inhibitory factor. The protocol can also be applied to efficiently derive mESC lines from non-permissive mouse strains, by the simple addition of a cocktail of two small-molecule inhibitors to the derivation medium (2i medium). Detailed procedures on the preparation and culture of feeder cells, collection and culture of mouse embryos, and derivation and culture of mESC lines are provided. This protocol does not require specialized equipment and can be carried out in any laboratory with basic mammalian cell culture expertise.

This article describes a protocol to efficiently derive and culture pluripotent stem cell lines from mouse embryos at the blastocyst stage.

Mouse embryonic stem cell (mESC) derivation is the process by which pluripotent cell lines are established from preimplantation embryos. These lines ...

Generation of Organoids from Mouse Extrahepatic Bile Ducts

Cholangiopathies, which affect extrahepatic bile ducts (EHBDs), include biliary atresia, primary sclerosing cholangitis, and cholangiocarcinoma. They have no effective therapeutic options. Tools to study EHBD are very limited. Our purpose was to develop an organ-specific, versatile, adult stem cell-derived, preclinical cholangiocyte model that can be easily generated from wild type and genetically engineered mice. Thus, we report on the novel technique of developing an EHBD organoid (EHBDO) culture system from adult mouse EHBDs. The model is cost-efficient, able to be readily analyzed, and has multiple downstream applications. Specifically, we describe the methodology of mouse EHBD isolation and single cell dissociation, organoid culture initiation, propagation, and long-term maintenance and storage. This manuscript also describes EHBDO processing for immunohistochemistry, fluorescent microscopy, and mRNA abundance quantitation by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). This protocol has significant advantages in addition to producing EHBD-specific organoids. The use of a conditioned medium from L-WRN cells significantly reduces the cost of this model. The use of mouse EHBDs provides almost unlimited tissue for culture generation, unlike human tissue. Generated mouse EHBDOs contain a pure population of epithelial cells with markers of endodermal progenitor and differentiated biliary cells. Cultured organoids maintain homogenous morphology through multiple passages and can be recovered after a long-term storage period in liquid nitrogen. The model allows for the study of biliary progenitor cell proliferation, can be manipulated pharmacologically, and may be generated from genetically engineered mice. Future studies are needed to optimize culture conditions in order to increase plating efficiency, evaluate functional cell maturity, and direct cell differentiation. Development of co-culture models and a more biologically neutral extracellular matrix are also desirable.

This protocol describes the production of a mouse extrahepatic bile duct 3-dimensional organoid system. These biliary organoids can be maintained in culture to study cholangiocyte biology. Biliary organoids express markers of both progenitor and biliary cells and are composed of polarized epithelial cells.

Cholangiopathies, which affect extrahepatic bile ducts (EHBDs), include biliary atresia, primary sclerosing cholangitis, and cholangiocarcinoma. They have ...

Imaging Dpp Release from a Drosophila Wing Disc

The transforming Growth Factor-beta (TGF-&#946;) superfamily is essential for early embryonic patterning and development of adult structures in multicellular organisms. The TGF-&#946; superfamily includes TGF-&#946;, bone morphogenetic protein (BMPs), Activins, Growth and Differentiation Factors, and Nodals. It has long been known that the amount of ligand exposed to cells is important for its effects. It was thought that long-range concentration gradients set up embryonic pattern. However, recently it has become clear that the timing of exposure to these ligands is also important for their downstream transcriptional consequences. A TGF-&#946; superfamily ligand cannot have a developmental consequence until it is released from the cell in which it was produced. Until recently, it was difficult to determine when these ligands were released from cells. Here we show how to measure the release of a Drosophila BMP called Decapentaplegic (Dpp) from the cells of the wing primordium or wing disc. This method could be modified for other systems or signaling ligands.

Imaging Dpp Release from a Drosophila Wing Disc

The timing of exposure to ligands may impact their developmental consequences. Here we show how to image release of a Drosophila bone morphogenetic protein (BMP) called Dpp from cells of the wing disc.

The transforming Growth Factor-beta (TGF-&#946;) superfamily is essential for early embryonic patterning and development of adult structures in ...

Translating Ribosome Affinity Purification (TRAP) to Investigate Arabidopsis thaliana Root Development at a Cell Type-Specific Scale

In this article, we give hands-on instructions to obtain translatome data from different Arabidopsis thaliana root cell types via the translating ribosome affinity purification (TRAP) method and consecutive optimized low-input library preparation.
As starting material, we employ plant lines that express GFP-tagged ribosomal protein RPL18 in a cell type-specific manner by use of adequate promoters. Prior to immunopurification and RNA extraction, the tissue is snap frozen, which preserves tissue integrity and simultaneously allows execution of time series studies with high temporal resolution. Notably, cell wall structures remain intact, which is a major drawback in alternative procedures such as fluorescence-activated cell sorting-based approaches that rely on tissue protoplasting to isolate distinct cell populations. Additionally, no tissue fixation is necessary as in laser capture microdissection-based techniques, which allows high-quality RNA to be obtained.
However, sampling from subpopulations of cells and only isolating polysome-associated RNA severely limits RNA yields. It is, therefore, necessary to apply sufficiently sensitive library preparation methods for successful data acquisition by RNA-seq.
TRAP offers an ideal tool for plant research as many developmental processes involve cell wall-related and mechanical signaling pathways. The use of promoters to target specific cell populations is bridging the gap between organ and single-cell level that in turn suffer from little resolution or very high costs. Here, we apply TRAP to study cell-cell communication in lateral root formation.

Translating Ribosome Affinity Purification TRAP to Investigate Arabidopsis thaliana Root Development at a Cell Type-Specific Scale

Translating ribosome affinity purification (TRAP) offers the possibility to dissect developmental programs with minimal processing of organs and tissues. The protocol yields high-quality RNA from cells targeted with a green fluorescent protein (GFP)-labeled ribosomal subunit. Downstream analysis tools, such as qRT-PCR or RNA-seq, reveal tissue and cell type-specific expression profiles.

In this article, we give hands-on instructions to obtain translatome data from different Arabidopsis thaliana root cell types via the translating ribosome ...

Generation of Na&#239;ve Blastoderm Explants from Zebrafish Embryos

Due to their optical clarity and rapid development, zebrafish embryos are an excellent system for examining cell behaviors and developmental processes. However, because of the complexity and redundancy of embryonic signals, it can be challenging to discern the complete role of any single signal during early embryogenesis. By explanting the animal region of the zebrafish blastoderm, relatively na&#239;ve clusters of embryonic cells are generated that can be easily cultured and manipulated ex vivo. By introducing a gene of interest by RNA injection before explantation, one can assess the effect of this molecule on gene expression, cell behaviors, and other developmental processes in relative isolation. Furthermore, cells from embryos of different genotypes or conditions can be combined in a single chimeric explant to examine cell/tissue interactions and tissue-specific gene functions. This article provides instructions for generating zebrafish blastoderm explants and demonstrates that a single signaling molecule - a Nodal ligand - is sufficient to induce germ layer formation and extension morphogenesis in otherwise na&#239;ve embryonic tissues. Due to their ability to recapitulate embryonic cell behaviors, morphogen gradients, and gene expression patterns in a simplified ex vivo system, these explants are anticipated to be of great utility to many zebrafish researchers.

Zebrafish blastoderm explants are generated by isolating embryonic cells from endogenous signaling centers within the early embryo, producing relatively na&#239;ve cell clusters easily manipulated and cultured ex vivo. This article provides instructions for making such explants and demonstrates their utility by interrogating roles for Nodal signaling during gastrulation.

Due to their optical clarity and rapid development, zebrafish embryos are an excellent system for examining cell behaviors and developmental processes. ...

Methods Article

Polysome Purification from Soybean Symbiotic Nodules