We thank Royena Tanaz, Huda Yousuf, and Sadiqa Taasen for technical help. We are very grateful to Prof. James Shorter for the generous provision of reagents and intellectual assistance in the design of sucrose tuning experiments. Yeast plasmids were a generous gift from Prof. Aaron Gitler (including 303Gal-FUS; Addgene plasmid # 29614). Brooklyn College and the Advanced Science Research Center (CUNY) as well as an NIH NINDS Advanced Postdoctoral Transition Award (K22NS09131401) supported M.P.T.

1. Transforming S. cerevisiae with neurodegenerative proteinopathy-associated protein constructs
<ol>
	<li>Grow wild type (WT) 303 yeast in yeast extract peptone dextrose (YPD) broth overnight with shaking (200 rpm) at 30 &#176;C.</li>
	<li>After 12&#8722;16 h of growth, dilute yeast to an optical density at 600 nm (OD600) of 0.25 with YPD. As 10 mL of yeast liquid culture will be needed for each transformation, prepare 50 mL of yeast liquid culture for five transformations corresponding to FUS, TDP-43, a-synuclein, and vector only (ccdB) constructs, as well as a negative control transformation without DNA.</li>
	<li>Grow yeast with agitation at 30 &#176;C until an OD600 between 0.60 and 0.80 is reached. This generally takes 4&#8722;6 h.</li>
	<li>Thirty minutes before yeast growth is complete, linearize plasmid constructs. 
	NOTE: The plasmid constructs used here must be integrated directly into the genome, and hence linearization is required prior to transformation.
	<ol>
		<li>Calculate the volume of plasmid stock that amounts to 1 &#181;g. Subtract this volume from 44 &#181;L to calculate the amount of nuclease free H2O needed.</li>
		<li>Add nuclease free H2O, 5 &#181;L of 10x restriction enzyme buffer (Table of Materials), 1 &#181;L of NheI restriction enzyme (Table of Materials), and the appropriate amount of plasmid (calculated in step 1.4.1) to a microcentrifuge tube. Mix thoroughly by pipetting up and down and incubate at 37 &#176;C for 15 min. The plasmid is now linearized and ready for transformation.</li>
	</ol>
	</li>
	<li>After yeast culture reaches an OD600 of 0.6-0.8, harvest cells in a 50 mL conical tube and spin down at 850 x g at 4 &#176;C for 3 min. Discard supernatant.</li>
	<li>Wash cell pellet in 10 mL of sterile H2O and centrifuge at 850 x g at 4 &#176;C for 3 min. Discard supernatant. Repeat 3x for a total of four washes.</li>
	<li>At the start of the third wash, thaw and boil 10 &#181;L of salmon sperm DNA per transformation reaction for 5 min on a heating block.</li>
	<li>Resuspend cell pellet in 100 &#181;L of dH2O per transformation and split into appropriate number of microcentrifuge tubes. For five transformations, resuspend pellet into 500 &#181;L of dH2O and split evenly into five separate microcentrifuge tubes.</li>
	<li>Centrifuge for 5 min at 850 x g at room temperature (RT) and remove all remaining water.</li>
	<li>Add, in the following order, 50 &#181;L of sterile H2O, 240 &#181;L of 50% polyethylene glycol (PEG), 36 &#181;L of 1 M LiAc, 10 &#181;L of salmon sperm DNA, 20 &#181;L of linearized plasmid DNA (or nuclease free water for no DNA control transformation). Mix thoroughly after each addition and vortex briefly after all transformation components have been added.</li>
	<li>Incubate transformation reactions at 42 &#176;C for 20 min. 
	NOTE: Carry out this incubation in a water bath for more consistent temperature control.</li>
	<li>Centrifuge at 470 x g at RT for 5 min. Discard supernatant and resuspend each cell pellet in 200 &#181;L of sterile H2O.</li>
	<li>Plate yeast suspension on selective media plates and spread with rolling beads or spreader. Incubate plates for 2&#8722;3 days at 30 &#176;C. 
	NOTE: Synthetic-defined (SD)-His plates are used for the plasmids described here.</li>
</ol>
2. Surveying colony growth suppression and storage in glycerol stocks
<ol>
	<li>Inoculate single colonies from transformation plates in 5 mL of selective media supplemented with 2% raffinose. Grow on a shaker at 30 &#176;C overnight. Select at least 12 colonies per transformation. 
	NOTE: No colonies are expected in the &#8220;no DNA&#8221; transformation control plate.</li>
	<li>Sterilize pin-frogger with ethanol, followed by flaming.</li>
	<li>For each culture, aliquot 100 &#181;L of saturated cell suspension in the first row of a 96-well plate. Add 200 &#181;L of sterile H2O in all wells of the adjacent well columns. For 1:5 serial dilutions, add 50 &#181;L from the first column to the adjacent column behind with multichannel pipet. Mix thoroughly by pipetting. Then add 50 &#181;L from the second column to the third column and mix by pipetting. Repeat this for the rest of the plate.</li>
	<li>Plate yeast by placing frogger in a 96-well plate and then stamping by gently and evenly pressing down on selective media plates with and without galactose. Incubate at 30 &#176;C for 2&#8722;3 days. 
	NOTE: SD-His and SGal-His plates are used for the plasmids described here.</li>
	<li>For FUS, TDP-43, and a-synuclein transformations, identify the colony displaying the most growth suppression in the presence of galactose. Select this colony from the SD-His plate and inoculate into 10 mL of selective media supplemented with 2% raffinose for overnight growth. For the vector control, pick a colony displaying no growth suppression in the presence of galactose.</li>
	<li>To prepare glycerol stocks, combine 0.5 mL of saturated liquid culture with 0.5 mL of 50% glycerol. Mix by pipetting up and down and freeze at -80 &#176;C. 
	NOTE: Glycerol stocks can be stored for up to a year at -80 &#176;C.</li>
</ol>
3. Overexpression of neurodegenerative proteinopathy associated proteins in S. cerevisiae
<ol>
	<li>From frozen glycerol stocks, re-streak yeast on histidine, 2% glucose agar selective media plates and incubate at 30 &#176;C for 2&#8722;3 days.</li>
	<li>Inoculate single colonies for each of overexpression models and control in 5 mL of histidine selective liquid media supplemented with 2% raffinose. Grow with shaking (200 rpm) at 30 &#176;C overnight.</li>
	<li>Grow 100 mL of liquid culture for each of the overexpression models and controls (FUS, TDP-43, and vector control; a-synuclein and vector control).
	<ol>
		<li>Prepare 100 mL of culture in selective media supplemented with 2% galactose.</li>
		<li>Measure the OD600 of overnight cultures and calculate the amount of overnight culture needed to render overexpression cultures at a starting OD600 of 0.3. Typically, overnight cultures will reach an OD600 of 0.9&#8722;10, requiring about 5 mL of overnight culture to start 100 mL of fresh culture.</li>
		<li>Induce protein overexpression by growing yeast on galactose media (see step 3.3.1) for 5 h (FUS and TDP-43) or 8 h (a-synuclein) with shaking at 30 &#176;C.</li>
	</ol>
	</li>
	<li>Measure OD600 of each culture at the end of the induction period. Standardize all cell counts to the lowest OD600 value. Harvest culture in 50 mL tubes by centrifuging at 850 x g for 5 min at 4 &#176;C. Discard supernatant. 
	NOTE: If the OD600 values measured at the end of induction are 0.654 and 0.984, respectively, for 100 mL of a-synuclein culture and 100 mL of control culture, harvest 95 mL of the a-synuclein culture and 67.1 mL of the control culture.</li>
	<li>Resuspend pellet in 1 mL of sterile dH2O for every 10 mL of culture grown. Split pellet evenly by aliquoting 1 mL of cell resuspension into microcentrifuge tubes. For example, if starting with 100 mL of culture, resuspend pellet in 10 mL of sterile dH2O and divide it into 10 microcentrifuge tubes.</li>
	<li>Centrifuge at 850 x g for 5 min at 4 &#176;C, then remove supernatant. Snap freeze cell pellets in liquid nitrogen and store at -80 &#176;C. 
	NOTE: Cell pellets can be stored at -80 &#176;C for up to one year.</li>
</ol>
4. Cell lysis and western blotting to detect histone post-translational modifications
<ol>
	<li>Cell lysis
	<ol>
		<li>Thaw yeast cell pellets on ice and resuspend cell pellets in 100 &#181;L of dH2O.</li>
		<li>To the cell resuspension, add 300 &#181;L of 0.2 M NaOH and 20 &#181;L of 2-mercaptoethanol. Resuspend by pipetting up and down.</li>
		<li>Incubate cells on ice for 10 min and then centrifuge at 3,200 x g on a tabletop centrifuge for 30 s at RT. Discard supernatant.</li>
		<li>Resuspend cell pellet in 100 &#181;L of 1x loading dye and boil for 10 min on a heating block. 
		NOTE: The recipe for 6x loading dye can be found in the Table of Materials.</li>
	</ol>
	</li>
	<li>Sodium dodecyl sulfate polyacrylamide gel electrophoresis and membrane transfer
	<ol>
		<li>Prepare gel chamber by placing two gels in a gel holder and filling inner chamber to the top with running buffer and filling the outside chamber to the two-gel line mark.</li>
		<li>Load 15 &#181;L of sample from step 4.1.4 per well into a 10-well 12% polyacrylamide gel. For the protein ladder lane, load 5 &#181;L.</li>
		<li>Run gel for approximately 45 min at 150 V, or until loading dye front reaches the bottom of the gel.</li>
		<li>While the gel is running, prepare for membrane transfer by soaking fiber pads (two per gel) in transfer buffer (Table of Materials) and soaking polyvinylidene fluoride (PVDF) membrane in methanol. Rinse membrane in transfer buffer before transfer. 
		NOTE: The PVDF membrane should be cut to the size of the gel and use one PVDF membrane for every gel being transferred.</li>
		<li>Assemble, on semi-dry transfer apparatus cell, a transfer &#8216;sandwich&#8217;: from bottom electrode, place (1) presoaked fiber pad, (2) presoaked and rinsed PVDF membrane, (3) gel from step 4.2.3, and (4) a second presoaked fiber pad. 
		NOTE: Make sure to avoid air bubbles in the transfer &#8216;sandwich.&#8217; Gently roll &#8216;sandwich&#8217; with serological pipet to force out bubbles. Once the gel has been placed on top of the PDVF membrane, make sure not to move it.</li>
		<li>Carry out protein transfer by setting power pack to 150 mA for 1 h (for one &#8216;sandwich&#8217;).</li>
	</ol>
	</li>
	<li>Detection of histone PTMs with modification specific antibodies
	<ol>
		<li>Remove membrane from transfer apparatus. Rinse briefly with dH2O.
		<ol>
			<li>Optionally, check transfer of proteins with Ponceau-S stain by pouring enough Ponceau-S stain to cover membrane in a small plastic box and incubating at RT with gentle shaking for 30&#8722;60 s. Remove Ponceau-S stain and rinse with dH2O until background stain disappears and protein bands are visible to the naked eye. Continue to rinse with dH2O until all stain is removed from membrane.</li>
		</ol>
		</li>
		<li>Block membrane by incubating blot for 1 h at RT with gentle rocking with tris buffered saline (TBS) blocking buffer (Table of Materials) in a small staining box. Use enough blocking buffer to cover blot. 
		NOTE: Be careful to place membrane upright in the staining box so that the membrane side on which proteins lie is not facing down.</li>
		<li>Incubate blot in the staining box overnight with a histone modification-specific antibody reactive towards yeast at 4 &#176;C. Dilute the antibody in TBS blocking buffer according to manufacturer specifications. Also include a proper nuclear loading control, such as anti-total H3 raised in a different host species from the modification-specific antibody. For instance, if probing for H3S10ph with an anti-H3S10ph antibody raised in rabbit, use an anti-total H3 antibody raised in mouse as a loading control. Repeat this for every blot as necessary. 
		NOTE: Antibody dilutions can be reused for a total of three times within a month. Store them at 4 &#176;C.</li>
		<li>Wash the membrane 4x in house-made TBS + 0.1% polysorbate 20 (TBST) for 5 min with rocking at RT.</li>
		<li>Incubate blot with fluorescent secondary antibodies at manufacturer-specified dilutions (donkey anti-rabbit 680 and donkey anti-mouse) for 1 h at RT. 
		NOTE: Fluorescent secondary antibodies must be protected from light during both storage and usage. Carry out incubation in dark plastic boxes or cover with aluminum foil.</li>
		<li>Wash membrane 4x with TBST for 5 min and wash with TBS for 5 min while rocking at RT.</li>
		<li>Visualize blot on a fluorescent Western blot imaging system for 2 min. Visualize the anti-rabbit 680 and anti-mouse 800 secondary antibodies in the 800 nm and 700 nm channels, respectively. 
		NOTE: Replicates are independently conducted starting from section 1. It is necessary to verify that the antibody signal response is within the range over which the signal response is linear for appropriate data interpretation in these experiments.</li>
	</ol>
	</li>
</ol>
5. Data analysis and statistics
<ol>
	<li>Open image of blot in an imaging software (Table of Materials). Acquire the raw density of modification-specific bands in the vector control, TDP-43, and FUS (or vector control and a-synuclein) samples, by drawing a rectangle that frames the band in analysis mode.</li>
	<li>Repeat step 5.1 for loading control bands. 
	NOTE: There will be a loading control band and a modification-specific band in each sample.</li>
	<li>Calculate the relative density of the modification-specific bands by dividing each band by the density of corresponding band in the vector control sample. This normalizes the data to the vector control.</li>
	<li>Calculate the relative density of the loading control band by dividing each band by the density of corresponding loading control band in the vector control sample.</li>
	<li>Calculate the adjusted relative density of each band by dividing the relative density of the modification-specific band over the relative density of the loading control band for each sample. Now, the data can be visualized in histogram form. 
	NOTE: For ease of processing, steps 5.3&#8211;5.5 can be done in a spreadsheet (Supplemental File).</li>
	<li>After multiple independent replicates, run Welch&#8217;s T-tests between the two control samples and the appropriate proteinopathy model (control versus FUS, control versus TDP-43, and control versus a-synuclein) with p = 0.05 as the cutoff for significance.</li>
</ol>

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S., Johnston, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+systems+of+glucose+repression+of+the+GAL1+promoter+in+Saccharomyces+cerevisiae.">Two systems of glucose repression of the GAL1 promoter in Saccharomyces cerevisiae.</a> Molecular and Cellular Biology. 10 (9), 4757 (1990).</li><li>Fernandez-Santiago, R., Ezquerra, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetic+Research+of+Neurodegenerative+Disorders+Using+Patient+iPSC-Based+Models.">Epigenetic Research of Neurodegenerative Disorders Using Patient iPSC-Based Models.</a> Stem Cells International. 2016, 9464591 (2016).</li><li>Armakola, 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=Inhibition+of+RNA+lariat+debranching+enzyme+suppresses+TDP-43+toxicity+in+ALS+disease+models.">Inhibition of RNA lariat debranching enzyme suppresses TDP-43 toxicity in ALS disease models.</a> Nature Genetics. 44 (12), 1302-1309 (2012).</li><li>Tibshirani, 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=Cytoplasmic+sequestration+of+FUS/TLS+associated+with+ALS+alters+histone+marks+through+loss+of+nuclear+protein+arginine+methyltransferase+1.">Cytoplasmic sequestration of FUS/TLS associated with ALS alters histone marks through loss of nuclear protein arginine methyltransferase 1.</a> Human Molecular Genetics. 24 (3), 773-786 (2015).</li><li>Masala, 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=Epigenetic+Changes+Associated+with+the+Expression+of+Amyotrophic+Lateral+Sclerosis+(ALS)+Causing+Genes.">Epigenetic Changes Associated with the Expression of Amyotrophic Lateral Sclerosis (ALS) Causing Genes.</a> Neuroscience. 390, 1-11 (2018).</li><li>Eryilmaz, I. 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=Epigenetic+approach+to+early-onset+Parkinson&#8217;s+disease:+low+methylation+status+of+SNCA+and+PARK2+promoter+regions.">Epigenetic approach to early-onset Parkinson&#8217;s disease: low methylation status of SNCA and PARK2 promoter regions.</a> Neurological Research. 39 (11), 965-972 (2017).</li><li>Daniele, S., 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=Epigenetic+Modifications+of+the+&#945;-Synuclein+Gene+and+Relative+Protein+Content+Are+Affected+by+Ageing+and+Physical+Exercise+in+Blood+from+Healthy+Subjects.">Epigenetic Modifications of the &#945;-Synuclein Gene and Relative Protein Content Are Affected by Ageing and Physical Exercise in Blood from Healthy Subjects.</a> Oxidative Medicine and Cellular Longevity. 2018, 3740345 (2018).</li></ol>

The authors have nothing to disclose.

The protocol described here provides a straightforward, expedient, and cost-effective way of categorizing genome-wide histone PTM changes correlated with neurodegenerative proteinopathies. While there are other models of ALS and PD, such as in vitro human cell lines and murine models32, S. cerevisiae remains attractive because of its ease of use. For instance, yeast models do not require use of a sterile hood, nor do they require the intensive training that goes along with cell c...

Neurodegenerative diseases are devastating illnesses with little to no treatment options available. Among these, amyotrophic lateral sclerosis (ALS) and Parkinson&#39;s disease (PD) are particularly dreadful. Approximately 90% of ALS and PD cases are considered sporadic, occurring without family history of the disease, while the remaining cases run in families and are generally linked to a specific gene mutation1,2. Interestingly, both of these diseases are associated with protein mislocalization and aggregation3,4,5,6. For instance, fused in sarcoma (FUS) and TAR DNA-binding protein 43 (TDP-43) are RNA binding proteins that mislocalize to the cytoplasm and aggregate in ALS7,8,9,10,11,12, while &#945;-synuclein is the principle component of proteinaceous aggregates termed Lewy bodies in PD5,13,14,15.
Despite the extensive genome-wide association efforts in large patient populations, the overwhelming majority of ALS and PD cases remain unexplained genetically. Can epigenetics play a role in neurodegenerative disease? Epigenetics comprises changes in gene expression occurring without changes to underlying DNA sequence16. A main epigenetic mechanism involves the post translational modifications (PTMs) of histone proteins16. In eukaryotic cells, genetic material is tightly wrapped into chromatin. The base unit of chromatin is the nucleosome, consisting of 146 base pairs of DNA wrapped around a histone octamer, composed of four pairs of histones (two copies each of histones H2A, H2B, H3, and H4)17. Each histone has an N-terminal tail that protrudes out of the nucleosome and can be modified by the addition of various chemical moieties, usually on lysine and arginine residues18. These PTMs are dynamic, which means they can be easily added and removed, and include groups such as acetylation, methylation, and phosphorylation. PTMs control the accessibility of DNA to the transcriptional machinery, and thus help control gene expression18. For example, histone acetylation reduces the strength of the electrostatic interaction between the highly basic histone protein and the negatively charged DNA backbone, allowing the genes packed by acetylated histones to be more accessible and thus highly expressed19. More recently, the remarkable biological specificity of particular histone PTMs and their combinations has led to the histone code hypothesis20,21 in which proteins that write, erase, and read histone PTMs all act in concert to modulate gene expression.
Yeast is a very useful model to study neurodegeneration. Importantly, many neuronal cellular pathways are conserved from yeast to humans22,23,24. Yeast recapitulate cytotoxicity phenotypes and protein inclusions upon overexpression of FUS, TDP-43, or &#945;-synuclein22,23,24,25,26. In fact, Saccharomyces cerevisiae models of ALS have been used to identify genetic risk factors in humans27. Furthermore, yeast overexpressing human &#945;-synuclein allowed for the characterization of the Rsp5 network as a druggable target to ameliorate &#945;-synuclein toxicity in neurons28,29.
Here, we describe a protocol exploiting Saccharomyces cerevisiae to detect genome-wide histone PTM changes associated with neurodegenerative proteinopathies (Figure 1). The use of S. cerevisiae is highly attractive because of its ease of use, low cost, and speed compared to other in vitro and animal models of neurodegeneration. Harnessing previously developed ALS and PD models22,23,25,26, we have overexpressed human FUS, TDP-43, and &#945;-synuclein in yeast and uncovered distinct histone PTM changes occurring in connection with each proteinopathy30. The protocol that we describe here can be completed in less than two weeks from transformation to data analysis.

<table>
	<tbody>
		<tr>
			<td>-His DO Supplement</td>
			<td>Clontech</td>
			<td>630415</td>
			<td></td>
		</tr>
		<tr>
			<td>10x Running Buffer</td>
			<td></td>
			<td></td>
			<td>Mix: 141.65 g glycine (ThermoFisher BP381-1), 30.3 g Tizma base (Sigma-Aldrich T6066), 10 g sodium dodecyl sulfate (Sigma-Aldrich L3771), and 1 L deionized water, pH 8.8.</td>
		</tr>
		<tr>
			<td>12% Polyacrylamide Gels</td>
			<td>BIO-RAD</td>
			<td>456-1041</td>
			<td></td>
		</tr>
		<tr>
			<td>2-mercaptoethanol</td>
			<td>Sigma-Aldrich</td>
			<td>M3148</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-acetyl-Histone H3 (Lys14) Primary Antibody</td>
			<td>MilliporeSigma</td>
			<td>07-353 (Lot No. 2762291)</td>
			<td>Dilution: 1/1000</td>
		</tr>
		<tr>
			<td>Anti-acetyl-Histone H4 (Lys 16) Primary Antibody</td>
			<td>Abcam</td>
			<td>ab109463 (Lot No. GR187780)</td>
			<td>Dilution: 1/2000</td>
		</tr>
		<tr>
			<td>Anti-acetyl-Histone H4 (Lys12) Primary Antibody</td>
			<td>Abcam</td>
			<td>ab46983 (Lot No. GR71882)</td>
			<td>Dilution: 1/5000</td>
		</tr>
		<tr>
			<td>Anti-dimethyl-Histone H3 (Lys36) Primary Antibody</td>
			<td>Abcam</td>
			<td>ab9049 (Lot No. GR266894, GR3236147)</td>
			<td>Dilution: 1/1000</td>
		</tr>
		<tr>
			<td>Anti-Histone H3 Primary Antibody</td>
			<td>Abcam</td>
			<td>ab24834 (Lot No. GR236539, GR174196, GR3194335)</td>
			<td>Nuclear Loading Control; Dilution: 1/2000</td>
		</tr>
		<tr>
			<td>Anti-phospho-Histone H2B (Thr129) Primary Antibody</td>
			<td>Abcam</td>
			<td>ab188292 (Lot No. GR211874)</td>
			<td>Dilution: 1/1000</td>
		</tr>
		<tr>
			<td>Anti-phospho-Histone H3 (Ser10) Primary Antibody</td>
			<td>Abcam</td>
			<td>ab5176 (Lot No. GR264582, GR192662, GR3217296)</td>
			<td>Dilution: 1/1000</td>
		</tr>
		<tr>
			<td>BioPhotometer D30</td>
			<td>Eppendorf</td>
			<td>6133000010</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Culture Dish (100 x 20 mm)</td>
			<td>Eppendorf</td>
			<td>30702118</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Culture Plate, 96 well</td>
			<td>Eppendorf</td>
			<td>30730011</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge 5804/5804 R/5810/5810 R</td>
			<td>Eppendorf</td>
			<td>22625501</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey Anti-Mouse IRDye 800 CW</td>
			<td>LI-COR</td>
			<td>926-32212 (Lot No. C60301-05, C61116-02, C80108-05)</td>
			<td>Dilution: 1/5000</td>
		</tr>
		<tr>
			<td>Donkey Anti-Rabbit IRDye 860 RD</td>
			<td>LI-COR</td>
			<td>926-68073 (Lot No. C60217-06, C70323-06, C70601-05, C80116-07)</td>
			<td>Dilution: 1/2500</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Sigma-Aldrich</td>
			<td>E7023</td>
			<td></td>
		</tr>
		<tr>
			<td>Extra thick blot paper (filter paper)</td>
			<td>BIO-RAD</td>
			<td>1703968</td>
			<td></td>
		</tr>
		<tr>
			<td>Galactose</td>
			<td>Sigma-Aldrich</td>
			<td>G0750</td>
			<td>Prepare 20% w/v stock solution.</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G8270</td>
			<td>Prepare 20% w/v stock solution.</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma-Aldrich</td>
			<td>G5516</td>
			<td>Prepare 50 % w/v solution.</td>
		</tr>
		<tr>
			<td>Immobilon-FL Transfer Membranes</td>
			<td>MilliporeSigma</td>
			<td>IPFL00010</td>
			<td></td>
		</tr>
		<tr>
			<td>Lithium acetate dihydrate (LiAc)</td>
			<td>Sigma-Aldrich</td>
			<td>L4158</td>
			<td>Prepare a 1 M solution.</td>
		</tr>
		<tr>
			<td>Loading Dye</td>
			<td></td>
			<td></td>
			<td>Mix: 1.2 g sodium dodecyl sulfate, 6 mg bromophenol blue (Sigma-Aldrich B8026), 4.7 mL glycerol, 1.2 mL 0.5M Trizma base pH 6.8, 0.93 g DL-Dithiothreitol (Sigma-Aldrich D0632), and 2.1 mL deionized water.</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>ThermoFisher</td>
			<td>A412-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini-PROTEAN Tetra Vertical Electrophoeresis Cell</td>
			<td>BIO-RAD</td>
			<td>1658004</td>
			<td></td>
		</tr>
		<tr>
			<td>Multichannel pipet</td>
			<td>Eppendorf</td>
			<td>2231300045</td>
			<td></td>
		</tr>
		<tr>
			<td>NEB Restriction Enzyme Buffer 2.1, 10x</td>
			<td>New England Bio Labs</td>
			<td>102855-152</td>
			<td></td>
		</tr>
		<tr>
			<td>Nhe I Restriction Enzyme</td>
			<td>New England Bio Labs</td>
			<td>101228-710</td>
			<td></td>
		</tr>
		<tr>
			<td>Nuclease Free Water</td>
			<td>Qiagen</td>
			<td>129114</td>
			<td></td>
		</tr>
		<tr>
			<td>Odyssey Fc Imaging System</td>
			<td>LI-COR Biosciences</td>
			<td>2800-03</td>
			<td></td>
		</tr>
		<tr>
			<td>OmniTray Cell Culture Treated w/Lid Sterile, PS (86 x 128 mm)</td>
			<td>ThermoFisher</td>
			<td>165218</td>
			<td></td>
		</tr>
		<tr>
			<td>pAG303GAL-a-synuclein-GFP</td>
			<td>Gift from A. Gitler</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>pAG303GAL-ccdB</td>
			<td>Addgene</td>
			<td>14133</td>
			<td></td>
		</tr>
		<tr>
			<td>pAG303Gal-FUS</td>
			<td>Addgene</td>
			<td>29614</td>
			<td></td>
		</tr>
		<tr>
			<td>pAG303GAL-TDP-43</td>
			<td>Gift from A. Gitler</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Poly(ethylene glycol) (PEG)</td>
			<td>Sigma-Aldrich</td>
			<td>P4338</td>
			<td>Prepare a 50% w/v solution.</td>
		</tr>
		<tr>
			<td>Ponceau S Stain</td>
			<td>Sigma-Aldrich</td>
			<td>P3504</td>
			<td>Mix: 0.5 g 0.1% w/w Ponceau S dye, 5 mL 1% v/v acetic acid (Sigma-Aldrich 320099), and 500 mL deionized water.</td>
		</tr>
		<tr>
			<td>PowerPac Basic Power Supply</td>
			<td>BIO-RAD</td>
			<td>164-5050</td>
			<td></td>
		</tr>
		<tr>
			<td>Raffinose pentahydrate</td>
			<td>Sigma-Aldrich</td>
			<td>R7630</td>
			<td>Prepare 10% w/v stock solution.</td>
		</tr>
		<tr>
			<td>Salmon Sperm DNA</td>
			<td>Agilent Tech</td>
			<td>201190</td>
			<td></td>
		</tr>
		<tr>
			<td>SD-His plates</td>
			<td></td>
			<td></td>
			<td>Mix: 20 g Agar (Sigma-Aldrich A1296), 0.77 g -His DO supplement, 6.7 g yeast Nitrogen Base w/o amino acids (ThermoFisher 291920), and 900 mL deionized water.</td>
		</tr>
		<tr>
			<td>SGal-His plates</td>
			<td></td>
			<td></td>
			<td>Mix: 20 g Agar, 0.77 g -His DO supplement, 6.7 g yeast Nitrogen Base w/o amino acids, 100 mL galactose solution, and 900 mL deionized water.</td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulfate Loading Buffer</td>
			<td></td>
			<td></td>
			<td>Store at -20 oC. 6X, Mix: 1.2 g sodium dodecyl sulfate, 6 mg bromophenol blue, 0.93 g DL-Dithiothreitol, 2.1 mL deionized water, 4.7 mL glycerol, and 1.2 mL 0.5 M Trizma base, pH 6.8.</td>
		</tr>
		<tr>
			<td>Sodium hydroxide</td>
			<td>Sigma-Aldrich</td>
			<td>221465</td>
			<td>Prepare 0.2 M solution.</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma-Aldrich</td>
			<td>84097</td>
			<td>Prepare 20% w/v stock solution.</td>
		</tr>
		<tr>
			<td>TBS + 0.1% Tween 20 (TBST)</td>
			<td></td>
			<td></td>
			<td>Mix: 100 mL 10X TBS, 1 mL Tween 20 (Sigma-Aldrich P7949), and 900 mL deionized water.</td>
		</tr>
		<tr>
			<td>TBS Blocking Buffer</td>
			<td>LI-COR</td>
			<td>927-5000</td>
			<td></td>
		</tr>
		<tr>
			<td>Trans-Blot SD Semi-Dry Electrophoretic Transfer Cell</td>
			<td>BIO-RAD</td>
			<td>170-3940</td>
			<td></td>
		</tr>
		<tr>
			<td>Transfer Buffer</td>
			<td></td>
			<td></td>
			<td>Mix: 22.5 g glycine, 4.84 g Tizma base, 400 mL methanol, 1 g sodium dodecyl sulfate, and 1.6 L deionized water.</td>
		</tr>
		<tr>
			<td>Tris-Buffered Saline (TBS)</td>
			<td></td>
			<td></td>
			<td>10X, 7.6 pH, Solution: Mix 24 g Trizma base, and 88 g sodium chloride (Sigma-Aldrich S7653). Fill to 1 L with deionized water.</td>
		</tr>
		<tr>
			<td>WT 303 S. cerevisiae yeast</td>
			<td>Gift from J. Shorter</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast Extract Peptone Dextrose (YPD)</td>
			<td>Sigma-Aldrich</td>
			<td>Y1375</td>
			<td></td>
		</tr>
	</tbody>
</table>

characterizing histone post-translational modification alterations in yeast neurodegenerative proteinopathy models

Neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and Parkinson&#39;s disease (PD), cause the loss of hundreds of thousands of lives each year. Effective treatment options able to halt disease progression are lacking. Despite the extensive sequencing efforts in large patient populations, the majority of ALS and PD cases remain unexplained by genetic mutations alone. Epigenetics mechanisms, such as the post-translational modification of histone proteins, may be involved in neurodegenerative disease etiology and progression and lead to new targets for pharmaceutical intervention. Mammalian in vivo and in vitro models of ALS and PD are costly and often require prolonged and laborious experimental protocols. Here, we outline a practical, fast, and cost-effective approach to determining genome-wide alterations in histone modification levels using Saccharomyces cerevisiae as a model system. This protocol allows for comprehensive investigations into epigenetic changes connected to neurodegenerative proteinopathies that corroborate previous findings in different model systems while significantly expanding our knowledge of the neurodegenerative disease epigenome.

To illustrate this method, we will take advantage of recently published results30. WT human FUS and TDP-43 were overexpressed for 5 h, while WT &#945;-synuclein was overexpressed for 8 h. A ccdB construct was used as a vector negative control. Figure 2 shows growth suppression in solid and liquid cultures. Yeast was harvested as described and Western blotting with modification-specific antibodies was performed. Anti-total H3 was used a...

Watch this Scientific Journal Video about Characterizing Histone Post-translational Modification Alterations in Yeast Neurodegenerative Proteinopathy Models at JoVE.com

Characterizing Histone Post-translational Modification Alterations in Yeast Neurodegenerative Proteinopathy Models

This protocol outlines experimental procedures to characterize genome-wide changes in the levels of histone post-translational modifications (PTM) occurring in connection with the overexpression of proteins associated with ALS and Parkinson&#39;s disease in Saccharomyces cerevisiae models. After SDS-PAGE separation, individual histone PTM levels are detected with modification-specific antibodies via Western blotting.

Neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and Parkinson&#39;s disease (PD), cause the loss of hundreds of thousands of lives ...

characterizing-histone-post-translational-modification-alterations

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JoVE Journal

Genetics

7.4K Views.  Brooklyn College. This protocol outlines experimental procedures to characterize genome-wide changes in the levels of histone post-translational modifications (PTM) occurring in connection with the overexpression of proteins associated with ALS and Parkinson's disease in Saccharomyces cerevisiae models. After SDS-PAGE separation, individual histone PTM levels are detected with modification-specific antibodies via Western blotting.

Video: Characterizing Histone Post-translational Modification Alterations in Yeast Neurodegenerative Proteinopathy Models

Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

Forward genetic screening in model organisms is the workhorse to discover functionally important genes and pathways in many biological processes. In most mutagenesis-based screens, researchers have relied on the use of toxic chemicals, carcinogens, or irradiation, which requires designated equipment, safety setup, and/or disposal of hazardous materials. We have developed a simple approach to induce heritable mutations in C. elegans using germline-expressed histone-miniSOG, a light-inducible potent generator of reactive oxygen species. This mutagenesis method is free of toxic chemicals and requires minimal laboratory safety and waste management. The induced DNA modifications include single-nucleotide changes and small deletions, and complement those caused by classical chemical mutagenesis. This methodology can also be used to induce integration of extrachromosomal transgenes. Here, we provide the details of the LED setup and protocols for standard mutagenesis and transgene integration.

Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

Genetically-encoded histone-miniSOG induces genome-wide heritable mutations in a blue&#160;light-dependent manner. This mutagenesis method is simple, fast, free of toxic chemicals, and well-suited for forward genetic screening and transgene integration.

Forward genetic screening in model organisms is the workhorse to discover functionally important genes and pathways in many biological processes. In most ...

Detection of Copy Number Alterations Using Single Cell Sequencing

Detection of genomic changes at single cell resolution is important for characterizing genetic heterogeneity and evolution in normal tissues, cancers, and microbial populations. Traditional methods for assessing genetic heterogeneity have been limited by low resolution, low sensitivity, and/or low specificity. Single cell sequencing has emerged as a powerful tool for detecting genetic heterogeneity with high resolution, high sensitivity and, when appropriately analyzed, high specificity. Here we provide a protocol for the isolation, whole genome amplification, sequencing, and analysis of single cells. Our approach allows for the reliable identification of megabase-scale copy number variants in single cells. However, aspects of this protocol can also be applied to investigate other types of genetic alterations in single cells.

Single cell sequencing is an increasingly popular and accessible tool for addressing genomic changes at high resolution. We provide a protocol that uses single cell sequencing to identify copy number alterations in single cells.

Detection of genomic changes at single cell resolution is important for characterizing genetic heterogeneity and evolution in normal tissues, cancers, and ...

Targeted in Situ Mutagenesis of Histone Genes in Budding Yeast

We describe a PCR- and homologous recombination-based system for generating targeted mutations in histone genes in budding yeast cells. The resulting mutant alleles reside at their endogenous genomic sites and no exogenous DNA sequences are left in the genome following the procedure. Since in haploid yeast cells each of the four core histone proteins is encoded by two non-allelic genes with highly homologous open reading frames (ORFs), targeting mutagenesis specifically to one of two genes encoding a particular histone protein can be problematic. The strategy we describe here bypasses this problem by utilizing sequences outside, rather than within, the ORF of the target genes for the homologous recombination step. Another feature of this system is that the regions of DNA driving the homologous recombination steps can be made to be very extensive, thus increasing the likelihood of successful integration events. These features make this strategy particularly well-suited for histone gene mutagenesis, but can also be adapted for mutagenesis of other genes in the yeast genome.

Targeted in Situ Mutagenesis of Histone Genes in Budding Yeast

A strategy for generating mutations in histone genes at their endogenous location in Saccharomyces cerevisiae is presented.

We describe a PCR- and homologous recombination-based system for generating targeted mutations in histone genes in budding yeast cells. The resulting ...

Chromatin Immunoprecipitation (ChIP) of Histone Modifications from Saccharomyces cerevisiae

Histone post-translational modifications (PTMs), such as acetylation, methylation and phosphorylation, are dynamically regulated by a series of enzymes that add or remove these marks in response to signals received by the cell. These PTMS are key contributors to the regulation of processes such as gene expression control and DNA repair. Chromatin immunoprecipitation (chIP) has been an instrumental approach for dissecting the abundance and localization of many histone PTMs throughout the genome in response to diverse perturbations to the cell. Here, a versatile method for performing chIP of post-translationally modified histones from the budding yeast Saccharomyces cerevisiae (S. cerevisiae) is described. This method relies on crosslinking of proteins and DNA using formaldehyde treatment of yeast cultures, generation of yeast lysates by bead beating, solubilization of chromatin fragments by micrococcal nuclease, and immunoprecipitation of histone-DNA complexes. DNA associated with the histone mark of interest is purified and subjected to quantitative PCR analysis to evaluate its enrichment at multiple loci throughout the genome. Representative experiments probing the localization of the histone marks H3K4me2 and H4K16ac in wildtype and mutant yeast are discussed to demonstrate data analysis and interpretation. This method is suitable for a variety of histone PTMs and can be performed with different mutant strains or in the presence of diverse environmental stresses, making it an excellent tool for investigating changes in chromatin dynamics under different conditions.

Chromatin Immunoprecipitation ChIP of Histone Modifications from Saccharomyces cerevisiae

Here, we describe a protocol for chromatin immunoprecipitation of modified histones from the budding yeast Saccharomyces cerevisiae. Immunoprecipitated DNA is subsequently used for quantitative PCR to interrogate the abundance and localization of histone post-translational modifications throughout the genome.

Histone post-translational modifications (PTMs), such as acetylation, methylation and phosphorylation, are dynamically regulated by a series of enzymes ...

Gene-targeted Random Mutagenesis to Select Heterochromatin-destabilizing Proteasome Mutants in Fission Yeast

Random mutagenesis of a target gene is commonly used to identify mutations that yield the desired phenotype. Of the methods that may be used to achieve random mutagenesis, error-prone PCR is a convenient and efficient strategy for generating a diverse pool of mutants (i.e., a mutant library). Error-prone PCR is the method of choice when a researcher seeks to mutate a pre-defined region, such as the coding region of a gene while leaving other genomic regions unaffected. After the mutant library is amplified by error-prone PCR, it must be cloned into a suitable plasmid. The size of the library generated by error-prone PCR is constrained by the efficiency of the cloning step. However, in the fission yeast, Schizosaccharomyces pombe, the cloning step can be replaced by the use of a highly efficient one-step fusion PCR to generate constructs for transformation. Mutants of desired phenotypes may then be selected using appropriate reporters. Here, we describe this strategy in detail, taking as an example, a reporter inserted at centromeric heterochromatin.

This article describes a detailed methodology for random mutagenesis of a target gene in fission yeast. As an example, we target rpt4+, which encodes a subunit of the 19S proteasome, and screen for mutations that destabilize heterochromatin.

Random mutagenesis of a target gene is commonly used to identify mutations that yield the desired phenotype. Of the methods that may be used to achieve ...

A Fast and Quantitative Method for Post-translational Modification and Variant Enabled Mapping of Peptides to Genomes

Cross-talk between genes, transcripts, and proteins is the key to cellular responses; hence, analysis of molecular levels as distinct entities is slowly being extended to integrative studies to enhance the understanding of molecular dynamics within cells. Current tools for the visualization and integration of proteomics with other omics datasets are inadequate for large-scale studies. Furthermore, they only capture basic sequence identify, discarding post-translational modifications and quantitation. To address these issues, we developed PoGo to map peptides with associated post-translational modifications and quantification to reference genome annotation. In addition, the tool was developed to enable the mapping of peptides identified from customized sequence databases incorporating single amino acid variants. While PoGo is a command line tool, the graphical interface PoGoGUI enables non-bioinformatics researchers to easily map peptides to 25 species supported by Ensembl genome annotation. The generated output borrows file formats from the genomics field and, therefore, visualization is supported in most genome browsers. For large-scale studies, PoGo is supported by TrackHubGenerator to create web-accessible repositories of data mapped to genomes that also enable an easy sharing of proteogenomics data. With little effort, this tool can map millions of peptides to reference genomes within only a few minutes, outperforming other available sequence-identity based tools. This protocol demonstrates the best approaches for proteogenomics mapping through PoGo with publicly available datasets of quantitative and phosphoproteomics, as well as large-scale studies.

Here we present the proteogenomic tool PoGo and protocols for fast, quantitative, post-translational modification and variant enabled mapping of peptides identified through mass spectrometry onto reference genomes. This tool is of use to integrate and visualize proteogenomic and personal proteomic studies interfacing with orthogonal genomics data.

Cross-talk between genes, transcripts, and proteins is the key to cellular responses; hence, analysis of molecular levels as distinct entities is slowly ...

Single Molecule Fluorescence In Situ Hybridization (smFISH) Analysis in Budding Yeast Vegetative Growth and Meiosis

Single molecule fluorescence in situ hybridization (smFISH) is a powerful technique to study gene expression in single cells due to its ability to detect and count individual RNA molecules. Complementary to deep sequencing-based methods, smFISH provides information about the cell-to-cell variation in transcript abundance and the subcellular localization of a given RNA. Recently, we have used smFISH to study the expression of the gene&#160;NDC80&#160;during meiosis in budding yeast, in which two transcript isoforms exist and the short transcript isoform has its entire sequence shared with the long isoform. To confidently identify each transcript isoform, we optimized known smFISH protocols and obtained high consistency and quality of smFISH data for the samples acquired during budding yeast meiosis. Here, we describe this optimized protocol, the criteria that we use to determine whether high quality of smFISH data is obtained, and some tips for implementing this protocol in&#160;other yeast strains and growth conditions.

Single Molecule Fluorescence In Situ Hybridization smFISH Analysis in Budding Yeast Vegetative Growth and Meiosis

This single molecule fluorescence in situ hybridization protocol is optimized to quantify the number of RNA molecules in budding yeast during vegetative growth and meiosis.

Single molecule fluorescence in situ hybridization (smFISH) is a powerful technique to study gene expression in single cells due to its ability to detect ...

Mating-based Overexpression Library Screening in Yeast

Budding yeast has been widely used as a model in studying proteins associated with human diseases. Genome-wide genetic screening is a powerful tool commonly used in yeast studies. The expression of a number of neurodegenerative disease-associated proteins in yeast causes cytotoxicity and aggregate formation, recapitulating findings seen in patients with these disorders. Here, we describe a method for screening a yeast model of the Amyotrophic Lateral Sclerosis-associated protein FUS for modifiers of its toxicity. Instead of using transformation, this new screening platform relies on the mating of yeast to introduce an arrayed library of plasmids into the yeast model. The mating method has two clear advantages: first, it is highly efficient; second, the pre-transformed arrayed library of plasmids can be stored for long-term as a glycerol stock, and quickly applied to other screens without the labor-intensive step of transformation into the yeast model each time. We demonstrate how this method can successfully be used to screen for genes that modify the toxicity of FUS.

This article presents a mating-based method to facilitate overexpression screening in budding yeast using an arrayed plasmid library.

Budding yeast has been widely used as a model in studying proteins associated with human diseases. Genome-wide genetic screening is a powerful tool ...

A Deep-sequencing-assisted, Spontaneous Suppressor Screen in the Fission Yeast Schizosaccharomyces pombe

A genetic screen for mutant alleles that suppress phenotypic defects caused by a mutation is a powerful approach to identify genes that belong to closely related biochemical pathways. Previous methods such as the Synthetic Genetic Array (SGA) analysis, and random mutagenesis techniques using ultraviolet (UV) or chemicals like ethyl methanesulfonate (EMS) or N-ethyl-N- nitrosourea (ENU), have been widely used but are often costly and laborious. Also, these mutagen-based screening methods are frequently associated with severe side effects on the organism, inducing multiple mutations that add to the complexity of isolating the suppressors. Here, we present a simple and effective protocol to identify suppressor mutations in mutants which confer a growth defect in Schizosaccharomyces pombe. The fitness of cells with a growth deficiency in standard rich liquid media or synthetic liquid media can be monitored for recovery using an automated 96-well plate reader over an extended period. Once a cell acquires a suppressor mutation in the culture, its descendants outcompete those of the parental cells. The recovered cells that have a competitive growth advantage over the parental cells can then be isolated and backcrossed with the parental cells. The suppressor mutations are then identified using whole-genome sequencing. Using this approach, we have successfully isolated multiple suppressors that alleviate the severe growth defects caused by loss of Elf1, an AAA+ family ATPase that is important in nuclear mRNA transport and maintenance of genomic stability. There are currently over 400 genes in S. pombe with mutants conferring a growth defect. As many of these genes are uncharacterized, we propose that our method will hasten the identification of novel functional interactions with this user-friendly, high-throughput approach.

A Deep-sequencing-assisted, Spontaneous Suppressor Screen in the Fission Yeast Schizosaccharomyces pombe

We present a simple suppressor screen protocol in fission yeast. This method is efficient, mutagen-free, and&#160; selective&#160; for&#160; mutations that&#160; often&#160; occur at&#160; &#160;a&#160; single&#160; genomic&#160; locus.&#160; The protocol is suitable for isolating suppressors that alleviate growth defects in liquid culture that are caused by a mutation or a drug.

A genetic screen for mutant alleles that suppress phenotypic defects caused by a mutation is a powerful approach to identify genes that belong to closely ...

Characterizing Mutational Load and Clonal Composition of Human Blood

Hematopoietic stem and progenitor cells (HSPCs) gradually accumulate DNA mutations during a lifespan, which can contribute to age-associated diseases such as leukemia. Characterizing mutation accumulation can improve understanding of the etiology of age-associated diseases. Presented here is a method to catalogue somatic mutations in individual HSPCs, which is based on whole-genome sequencing (WGS) of clonal primary cell cultures. Mutations that are present in the original cell are shared by all cells in the clonal culture, whereas mutations acquired in vitro after cell sorting are present in a subset of cells. Therefore, this method allows for accurate detection of somatic mutations present in the genomes of individual HSPCs, which accumulate during life. These catalogues of somatic mutations can provide valuable insights into mutational processes active in the hematopoietic tissue and how these processes contribute to leukemogenesis. In addition, by assessing somatic mutations that are shared between multiple HSPCs of the same individual, clonal lineage relationships and population dynamics of blood populations can be determined. As this approach relies on in vitro expansion of single cells, the method is limited to hematopoietic cells with sufficient replicative potential.

Somatic mutation patterns in cells reflect previous mutagenic exposure and can reveal developmental lineage relationships. Presented here is a methodology to catalogue and analyze somatic mutations in individual hematopoietic stem and progenitor cells.

Hematopoietic stem and progenitor cells (HSPCs) gradually accumulate DNA mutations during a lifespan, which can contribute to age-associated diseases such ...