Name: Chromatin Accessibility Profiling in Whole Caenorhabditis elegans L4 Larvae (Video) | JoVE Journal
Uploaded: 2026-04-17T13:25:00.000+00:00
Duration: 8 min 2 s
Description: Learn ATAC-seq for chromatin accessibility profiling in whole Caenorhabditis elegans L4 larvae. This optimized protocol overcomes cuticle barriers and enables genome-wide epigenetic studies in one day.

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5 minutes

J Hersch-Gonza&#769;lez received a doctoral scholarship from the Consejo Nacional de Humanidades, Ciencias y Tecnologi&#769;as, CONAHCYT (now Secretari&#769;a de Ciencia, Humanidades, Tecnologi&#769;a e Innovacio&#769;n, SECIHTI) (#CVU 846476). J Hersch-Gonza&#769;lez was supported by a CONAHCYT (now SECIHTI) scholarship (788519). This project was supported by the PAPIIT-UNAM grant IN217824 and SECIHTI grant CBF-2025-I-2275 to VJV. At IFC, we thank the UBM: Laura Ongay-Larios, Guadalupe Co&#769;diz Huerta, and Minerva Mora Cabrera; the UBMI: Augusto Ce&#769;sar Poot-Herna&#769;ndez and Carlos Peralta Alvarez; and Unidad de Co&#769;mputo, Imagenologi&#769;a, Taller, and Biblioteca. We gratefully acknowledge the Caenorhabditis Genetics Center (CGC) for providing the strains used in this work. The CGC is funded by the NIH Office of Research Infrastructure Programs (P40 OD010440).

It is recommended to wear gloves at all times.
NOTE: Day 1: Preparing the synchronized worm population. All worms were grown on NGM plates seeded with E. coli OP50. Things needed before starting: (per sample or strain), One 90 mm or two 60 mm adult worm plate(s), One 90 mm or two 60 mm plates with food, M9 sterile buffer27 (3 g KH2PO4, 6 g Na2HPO4, 5 g NaCl, 1 ml 1 M MgSO4, H2O to 1 L. Sterilize by autoclaving)
1. Collection of worms
<ol>
	<li>Take a 90 mm (or two 30 mm) plate with the worm&#8217;s adult population.</li>
	<li>Recover the worms with M9 buffer and transfer them into a 15 mL centrifuge tube.</li>
	<li>Repeat the previous step to recover all the worms from the plate(s).</li>
	<li>Let the worms settle in the 15 mL tube for a minute and carefully remove the M9 buffer.</li>
	<li>Be sure to have ~1 mL of worms.</li>
</ol>
2. Bleaching
<ol>
	<li>Add 5 mL of M9 buffer + 650 &#181;L of NaOH 5M + 1,300 &#181;L Household bleach (5% solution of NaClO).</li>
	<li>Vortex at max. velocity for 1&#8211;2 min (not more than 2.5 min).</li>
	<li>Centrifugate at 1,000 x g for 1 min at RT (room temperature).</li>
	<li>Remove the supernatant and add 9 mL of M9 buffer.</li>
	<li>Repeat sections 2.3 and 2.4 five more times.</li>
	<li>Resuspend the embryos in 7 mL of M9 buffer. 
	NOTE: Bleaching protocol may vary in efficiency depending on temperature, type of commercial bleach28.</li>
</ol>
3. M9 (liquid) overnight hatching of the eggs
<ol>
	<li>Be sure that the collection tube with the bleached eggs is properly closed.</li>
	<li>Put the tube in slow movement at 20 &#176;C (e.g., in a shaker inside a room at 20 &#176;C).</li>
	<li>Worms will hatch overnight and arrest in L1. 
	NOTE: Don&#8217;t leave the tube for more than 12 h.</li>
</ol>
4. Day 2: growth of worms: recovering L1 worms.
<ol>
	<li>Take the tube from the shaker and corroborate under the microscope that there are L1 worms.</li>
	<li>Let the tube settle vertically for about 1 h at RT.</li>
	<li>Remove the supernatant and transfer ~300 L1 worms to one 90 mm or two 30 mm plates with food.</li>
	<li>Place them in a 20 &#176;C incubator and let them grow for 26 h. to reach the L4 stage.</li>
</ol>
5. Day 3: Preparation of stock solutions: The next solutions must be prepared to make fresh solutions on day 4
NOTE: This can be done on day 2 or 3.
<ol>
	<li>1 mL of 10% SDS.</li>
	<li>50 mL of 1M HEPES pH 8 (autoclave).</li>
	<li>250 mL of 20 % Sucrose (autoclave).</li>
	<li>100 mL of 2M NaCl: prepare (autoclave).</li>
	<li>50 mL of 2M KCl (autoclave).</li>
	<li>10 mL of 1M CaCl2 (autoclave).</li>
	<li>10 mL of 1M MgCl2 (autoclave).</li>
	<li>100 mL of 1M Tris, pH 7.4.</li>
	<li>5 mL of 5M NaCl (autoclave).</li>
	<li>500 &#181;L of 10% Tween-20.</li>
	<li>500 &#181;L of 10% Nonidet P40/IGEPAL.</li>
	<li>100 &#181;L of 5% Digitonin (needs to be heated to 65 &#730;C to dissolve).</li>
	<li>500 &#181;L of 10% BSA (filter with 0.22 &#181;m) stored at 4 &#730;C. 
	Day 4: L4 stage worms for ATAC 
	NOTE: Before starting, be sure to have an incubator at 25 &#730;C, a 4 &#730;C centrifuge ready, heat up Digitonin to 65 &#730;C (for fresh buffer preparation), have sterile M9 buffer, sterile H2O, and 20 &#181;m filters.</li>
</ol>
6. Solution preparation
<ol>
	<li>Prepare fresh solutions (see Table 1). Always handle L-15 10% FBS and egg buffer in sterile conditions and keep them cold.</li>
</ol>
Table 1: Stocks for fresh solution preparation. Recipes for stock preparation for nematode dissociation and cell suspension protocol (sections 6 and 7). <a href="https://www.jove.com/files/ftp_upload/69791/Table1_JoVE.xlsx" target="_blank">Please click here to download this Table.</a>
7. Nematode dissociation and cell suspension protocol
NOTE: One 90 mm or two 30 mm plates with an L4 synchronized worm population will be needed.
<ol>
	<li>After 26 h in the 20 &#176;C incubator, corroborate under the microscope that the majority of the worms are in the L4 stage.</li>
	<li>Recover all the worms from the plates with M9 buffer and put them into a 1.5 mL centrifuge tube.</li>
	<li>Wash 5 times with 1 mL of sterile H2O by letting the worms settle by gravity.</li>
	<li>Transfer the tube with the worms to a 25 &#176;C incubator for 30 min to get rid of intestinal food remains.</li>
	<li>Take 50 &#181;L of the worm pellet and place it into a new tube.</li>
	<li>Add 200 &#181;L of SDS-DTT fresh buffer and mix in a tube rotator for exactly 5 min (not longer).</li>
	<li>Add 1 mL of cold egg buffer and give a spin on a picofuge to remove supernatant. Repeat 6 times.</li>
	<li>Add 80 &#181;L of fresh prepared pronase (15 mg/mL).</li>
	<li>With a 200 &#181;L pipette (with filtered tips), mix 70 times the worms-pronase mix and incubate for 15 min at RT.</li>
	<li>Add 800 &#181;L of L-15 10% FBS media and centrifuge 5 min at 500 x g at 4 &#176;C.</li>
	<li>Add 1 mL of L-15 10% FBS media and repeat centrifugation.</li>
	<li>Repeat the previous step twice more.</li>
	<li>Remove supernatant and re-suspend in 900 &#181;L of L-15 10% FBS media.</li>
	<li>Filter with a 20 &#181;m filter.</li>
</ol>
8. Nuclei isolation and Chromatin tagmentation
NOTE: Use pipette filtered tips. Before starting: Take out Tn5 Buffer, let it thaw, set a thermoblock to 37 &#176;C, take out PCR product purification kit (Table of materials), put EB Buffer at 37 &#176;C, Centrifugation of the columns should be performed at RT.
<ol>
	<li>Centrifuge the filtered cell suspension for 5 min at 500 x g at 4 &#176;C.</li>
	<li>Remove supernatant, leaving ~50 &#181;L.</li>
	<li>Add 45 &#181;L of Lysis Buffer (freshly prepared) and carefully re-suspend.</li>
	<li>Carefully add 50 &#181;L of Wash Buffer (do not mix).</li>
	<li>Centrifuge for 5 min al 500 x g at 4 &#730;C. Remove supernatant. Keep it in the ice.</li>
	<li>While spinning, prepare for each reaction (Rx) the following Tn5 mix per sample: 25 &#181;L Tn5 Buffer29, 24.8 &#181;L Nuclease-free H2O, 0.2 &#181;L Tn5 enzyme. Prepare a master mix for multiple samples.</li>
	<li>After centrifugation, remove the supernatant. Keep the nuclei on ice. Add 50 &#181;L of Tn5 mix to the nuclei pellet and slowly pipette seven times. Incubate at 37 &#176;C for 30 min on a thermoblock. Make sure to give little taps to each tube every 10 min. 
	NOTE: Remove the PCR purification kit and place the EB Buffer in a 37 &#176;C incubator. The next 5 steps will use this Kit&#8217;s buffers.</li>
	<li>After Tn5 incubation, add 300 &#181;L of PB buffer (Yellow) from the kit and mix in a vortex at maximum speed for 25 s.</li>
	<li>Take all the volume from the tube and transfer it into the spin column (purple, stored at 4 &#176;C) and centrifuge 1 min at maximum speed at RT. 
	NOTE: The column will hold the DNA, so the supernatant can be trustingly discarded.</li>
	<li>Add 750 &#181;L of PE Buffer to the spin column and centrifuge 1 min at maximum speed at RT.</li>
	<li>Discard the supernatant with the collection tube. Place the column in a new 1.5 mL tube and repeat centrifugation; discard the 1.5 mL tube.</li>
	<li>Place the column in a new, labeled 1.5 mL tube and add 10 &#181;L of EB Buffer at 37 &#176;C to the center of the column. Be sure not to touch the column. Leave it at RT for 1 min.</li>
	<li>Centrifugate for 1 min at maximum speed at RT; never at 4 &#176;C.</li>
	<li>Repeat the last 2 steps for a total of 20 &#181;L elution in EB buffer. 
	NOTE: Optional pause: If stopping, sample(s) can be stored at -20 &#176;C. Here, the tagmented chromatin is in a 20 &#181;L volume.</li>
</ol>
9. Library amplification
<ol>
	<li>For ATAC library amplification, a real-time PCR has to be performed to estimate the proper cycle amplification number (see Table 2). Perform this on a PCR plate.</li>
	<li>To estimate the cycle numbers for final amplification, the cycle number that corresponds to the first third of the amplification curve has to be taken. It is ideal for the cycle number not to exceed 15. The cycle number must be estimated for each sample. 
	NOTE: After qPCR, the tagmented DNA is in a 18 &#181;L volume. Optional pause: If wanting to stop, samples can be stored at -20 &#730;C.</li>
	<li>ATAC Library final amplification:
	<ol>
		<li>Each sample must have a different Rv primer (take notes).</li>
		<li>Each sample must be amplified for the cycle number (N) estimated for each one.</li>
		<li>Perform this PCR in 200 &#181;L tubes (see Table 3).</li>
		<li>Here, there are 8 &#181;L of tagmented DNA and 50 &#181;L of amplified ATAC libraries</li>
		<li>Optional pause: If wanting to stop, sample(s) can be stored at -20 &#730;C.</li>
	</ol>
	</li>
</ol>
Table 2: Instructions for first library amplification. Recipe and PCR program for first library amplification (section 9). <a href="https://www.jove.com/files/ftp_upload/69791/Table2_JoVE.xlsx" target="_blank">Please click here to download this Table.</a>
Table 3: Instructions for ATAC Library final amplification. Recipe and PCR program for final library amplification (section 9). <a href="https://www.jove.com/files/ftp_upload/69791/Table3_JoVE.xlsx" target="_blank">Please click here to download this Table.</a>
10. Library purification
NOTE: Before starting, take the size selection beads (AMPure, SPRIselect, or homemade calibrated beads) out of the fridge to let them adjust to room temperature. Mix the beads vigorously using the vortex every time before using.
<ol>
	<li>Prepare 1 mL of 80% Ethanol (molecular grade).</li>
	<li>Add 25 &#181;L (0.5 vol) of beads to the sample tube (where there are 50 &#181;L) and mix with the pipette.</li>
	<li>Incubate at RT for 10 min.</li>
	<li>Place the tubes in the magnetic rack for 5 min.</li>
	<li>Transfer the supernatant (70 &#181;L) to a new clean 200 &#181;L tube. 
	NOTE: In this step, the &#60;200 bp DNA fragments are discarded.</li>
	<li>Add 65 &#181;L (1.3 vol) of beads and mix 10 times.</li>
	<li>Incubate at RT for 10 min.</li>
	<li>Place the tubes in the magnetic rack for 5 min.</li>
	<li>Discard the supernatant (without taking the tube from the magnetic rack). 
	NOTE: At this point, the beads are holding the &#62;200 bp and &#60;1000 bp DNA fragments.</li>
	<li>With the tube still on the magnetic rack, add 200 &#181;L of fresh prepared 80% ethanol directly over the beads and pipet up and down several times to wash them.</li>
	<li>Remove ethanol and repeat washing with 200 &#181;L of ethanol.</li>
	<li>Discard the ethanol, then let the beads dry with the lid open for ~2&#8211;5 min (don&#8217;t let them dry too much).</li>
	<li>Take the sample(s) out of the magnetic rack. Re-suspend in 50 &#181;L of sterile H2O. Make sure to wash all beads from the tube&#39;s walls.</li>
	<li>Finally, incubate the tube(s) in the magnetic rack for 5 min.</li>
	<li>Transfer the supernatant to a new clean tube. Here, the amplified and purified ATAC Libraries are stored. 
	NOTE: Libraries can be stored at -20 &#730;C for a short time (up to four weeks) or for longer periods at -80 &#730;C.</li>
</ol>
11. Fragment size distribution
<ol>
	<li>Once the final amplification is done, it is advisable to run a part of the sample through a capillary electrophoresis to be able to see the fragment size distribution pattern.</li>
</ol>

<ol><li>Poulet, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Identification+and+characterization+of+histones+in+Physarum+polycephalum+evidence+a+phylogenetic+vicinity+of+mycetozoans+to+the+animal+kingdom%5BTitle%5D)+AND+%22NAR+Genom.+Bioinform%22%5BJournal%5D)">Identification and characterization of histones in Physarum polycephalum evidence a phylogenetic vicinity of mycetozoans to the animal kingdom.</a> NAR Genom. Bioinform. 3 (4), lqab107(2021).</li>
<li>Meléndez-Ramírez, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dynamic+landscape+of+chromatin+accessibility+and+transcriptomic+changes+during+differentiation+of+human+embryonic+stem+cells+into+dopaminergic+neurons%5BTitle%5D)+AND+%22Sci.+Rep%22%5BJournal%5D)">Dynamic landscape of chromatin accessibility and transcriptomic changes during differentiation of human embryonic stem cells into dopaminergic neurons.</a> Sci. Rep. 11 (1), 16977(2021).</li>
<li>Mansisidor, A. R., Risca, V. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chromatin+accessibility%3A+methods%2C+mechanisms%2C+and+biological+insights%5BTitle%5D)+AND+%22Nucleus%22%5BJournal%5D)">Chromatin accessibility: methods, mechanisms, and biological insights.</a> Nucleus. 13 (1), 236-276 (2022).</li>
<li>Koyanagi, K. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Inferring+chromatin+accessibility+during+murine+hematopoiesis+through+phylogenetic+analysis%5BTitle%5D)+AND+%22BMC+Res.+Notes%22%5BJournal%5D)">Inferring chromatin accessibility during murine hematopoiesis through phylogenetic analysis.</a> BMC Res. Notes. 16 (1), 222(2023).</li>
<li>Gómora-García, J. C., Furlan-Magaril, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pioneer+factors+outline+chromatin+architecture%5BTitle%5D)+AND+%22Curr.+Opin.+Cell+Biol%22%5BJournal%5D)">Pioneer factors outline chromatin architecture.</a> Curr. Opin. Cell Biol. 93, 102480(2025).</li>
<li>Boivin, A., Dura, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vivo+chromatin+accessibility+correlates+with+gene+silencing+in+Drosophila%5BTitle%5D)+AND+%22Genetics%22%5BJournal%5D)">In vivo chromatin accessibility correlates with gene silencing in Drosophila.</a> Genetics. 150 (4), 1539-1549 (1998).</li>
<li>Penagos-Puig, A., Furlan-Magaril, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Heterochromatin+as+an+important+driver+of+genome+organization%5BTitle%5D)+AND+%22Front.+Cell+Dev.+Biol%22%5BJournal%5D)">Heterochromatin as an important driver of genome organization.</a> Front. Cell Dev. Biol. 8, 579137(2020).</li>
<li>Escamilla-Del-Arenal, M., Recillas-Targa, F. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((GATA-1+modulates+the+chromatin+structure+and+activity+of+the+chicken+alpha-globin+3%E2%80%B2+enhancer%5BTitle%5D)+AND+%22Mol.+Cell.+Biol%22%5BJournal%5D)">GATA-1 modulates the chromatin structure and activity of the chicken alpha-globin 3′ enhancer.</a> Mol. Cell. Biol. 28 (2), 575-586 (2008).</li>
<li>Zhong, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mapping+nucleosome+positions+using+DNase-seq%5BTitle%5D)+AND+%22Genome+Res%22%5BJournal%5D)">Mapping nucleosome positions using DNase-seq.</a> Genome Res. 26 (3), 351-364 (2016).</li>
<li>Johnson, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Flexibility+and+constraint+in+the+nucleosome+core+landscape+of+Caenorhabditis+elegans+chromatin%5BTitle%5D)+AND+%22Genome+Res%22%5BJournal%5D)">Flexibility and constraint in the nucleosome core landscape of Caenorhabditis elegans chromatin.</a> Genome Res. 16 (12), 1505-1516 (2006).</li>
<li>Boyle, A. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High-resolution+mapping+and+characterization+of+open+chromatin+across+the+genome%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">High-resolution mapping and characterization of open chromatin across the genome.</a> Cell. 132 (2), 311-322 (2008).</li>
<li>Buenrostro, J. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transposition+of+native+chromatin+for+fast+and+sensitive+epigenomic+profiling+of+open+chromatin%2C+DNA-binding+proteins%2C+and+nucleosome+position%5BTitle%5D)+AND+%22Nat.+Methods%22%5BJournal%5D)">Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins, and nucleosome position.</a> Nat. Methods. 10 (12), 1213-1218 (2013).</li>
<li>Buenrostro, J. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((ATAC-seq%3A+A+method+for+assaying+chromatin+accessibility+genome-wide%5BTitle%5D)+AND+%22Curr.+Protoc.+Mol.+Biol%22%5BJournal%5D)">ATAC-seq: A method for assaying chromatin accessibility genome-wide.</a> Curr. Protoc. Mol. Biol. 109, 21.29.1-21.29.9 (2015).</li>
<li>Corces, M. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+chromatin+accessibility+landscape+of+primary+human+cancers%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">The chromatin accessibility landscape of primary human cancers.</a> Science. 362 (6413), eaav1898(2018).</li>
<li>Shema, E., Bernstein, B. E., Buenrostro, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single-cell+and+single-molecule+epigenomics+to+uncover+genome+regulation+at+unprecedented+resolution%5BTitle%5D)+AND+%22Nat.+Genet%22%5BJournal%5D)">Single-cell and single-molecule epigenomics to uncover genome regulation at unprecedented resolution.</a> Nat. Genet. 51 (1), 19-25 (2019).</li>
<li>Cao, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+human+cell+atlas+of+fetal+gene+expression%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">A human cell atlas of fetal gene expression.</a> Science. 370 (6518), eaba7721(2020).</li>
<li>Ma, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chromatin+potential+identified+by+shared+single-cell+profiling+of+RNA+and+chromatin%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Chromatin potential identified by shared single-cell profiling of RNA and chromatin.</a> Cell. 183 (4), 1103-1116.e20 (2020).</li>
<li>Jänes, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chromatin+accessibility+dynamics+across+C.+elegans+development+and+ageing%5BTitle%5D)+AND+%22eLife%22%5BJournal%5D)">Chromatin accessibility dynamics across C. elegans development and ageing.</a> eLife. 7, e37344(2018).</li>
<li>Dhillon-Richardson, R. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fishing+for+developmental+regulatory+regions%3A+Zebrafish+tissue-specific+ATAC-seq%5BTitle%5D)+AND+%22Methods+Mol.+Biol%22%5BJournal%5D)">Fishing for developmental regulatory regions: Zebrafish tissue-specific ATAC-seq.</a> Methods Mol. Biol. 2599, 271-282 (2023).</li>
<li>Huynh, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genetic+variation+in+chromatin+state+across+multiple+tissues+in+Drosophila+melanogaster%5BTitle%5D)+AND+%22PLoS+Genet%22%5BJournal%5D)">Genetic variation in chromatin state across multiple tissues in Drosophila melanogaster.</a> PLoS Genet. 19 (5), e1010439(2023).</li>
<li>Lee, R. C., Feinbaum, R. L., Ambros, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+C.+elegans+heterochronic+gene+lin-4+encodes+small+RNAs+with+antisense+complementarity+to+lin-14%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14.</a> Cell. 75 (5), 843-854 (1993).</li>
<li>Fire, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Potent+and+specific+genetic+interference+by+double-stranded+RNA+in+Caenorhabditis+elegans%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans.</a> Nature. 391 (6669), 806-811 (1998).</li>
<li>Hillier, L. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genomics+in+C.+elegans%3A+so+many+genes%2C+such+a+little+worm%5BTitle%5D)+AND+%22Genome+Res%22%5BJournal%5D)">Genomics in C. elegans: so many genes, such a little worm.</a> Genome Res. 15 (12), 1651-1660 (2005).</li>
<li>Daugherty, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chromatin+accessibility+dynamics+reveal+novel+functional+enhancers+in+C.+elegans%5BTitle%5D)+AND+%22Genome+Res%22%5BJournal%5D)">Chromatin accessibility dynamics reveal novel functional enhancers in C. elegans.</a> Genome Res. 27 (12), 2096-2107 (2017).</li>
<li>Johnstone, I. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+cuticle+of+the+nematode+Caenorhabditis+elegans%3A+a+complex+collagen+structure%5BTitle%5D)+AND+%22Bioessays%22%5BJournal%5D)">The cuticle of the nematode Caenorhabditis elegans: a complex collagen structure.</a> Bioessays. 16 (3), 171-178 (1994).</li>
<li>Zhang, S., Banerjee, D., Kuhn, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+and+culture+of+larval+cells+from+C.+elegans%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Isolation and culture of larval cells from C. elegans.</a> PLoS One. 6 (4), e19505(2011).</li>
<li>Stiernagle, T. Maintenance of Caenorhabditis elegans. WormBook. , The C. elegans. Research Community. (2006).</li>
<li>Porta-de-la-Riva, M., Fontrodona, L., Villanueva, A., Cerón, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Basic+Caenorhabditis+elegans+methods%3A+synchronization+and+observation%5BTitle%5D)+AND+%22JoVE%22%5BJournal%5D)">Basic Caenorhabditis elegans methods: synchronization and observation.</a> JoVE. (64), e4019(2012).</li>
<li>Corces, M. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+improved+ATAC-seq+protocol+reduces+background+and+enables+interrogation+of+frozen+tissues%5BTitle%5D)+AND+%22Nat.+Methods%22%5BJournal%5D)">An improved ATAC-seq protocol reduces background and enables interrogation of frozen tissues.</a> Nat. Methods. 14 (10), 959-962 (2017).</li>
</ol>

The authors declare that they have no conflict of interest. No generative AI was used in the writing, analysis, or preparation of this manuscript.

Here we describe an optimized protocol for profiling genome-wide chromatin accessibility by ATAC-seq in whole L4-stage Caenorhabditis elegans worms. Chromatin accessibility directly influences gene expression, DNA repair, replication, and recombination, and while ATAC-seq has become a standard approach for cell and tissue samples, its application to intact organisms poses unique technical challenges, particularly in nematodes with an impermeable, collagen-rich cuticle. At the same time, assessing whole-organism ...

Eukaryotic genomes are densely packed inside the cell nucleus. To cope with spatial constraints, DNA is tightly wrapped around nucleosomes, multiprotein complexes composed of eight histones that are highly conserved across different phyla1. Chromatin accessibility patterns vary depending on the cellular state in a tissue, and gene-specific patterns2,3,4. Nucleosome accessibility strongly correlates with active transcription, particularly at promoters and distal regulatory regions such as enhancers, which are accessible to transcription factors5. Open chromatin regions also play important roles in DNA repair, replication, and chromosome segregation3. In contrast, reduced accessibility usually correlates with silenced heterochromatin and repetitive sequences6,7.
Mapping chromatin accessibility has been a powerful approach for understanding gene regulation. Different techniques have been employed to interrogate chromatin accessibility. Among the earliest methods, micrococcal nuclease (MNase) and DNase I digestion were used to map nucleosome positioning and accessibility at specific loci8,9. These techniques were later implemented for genome-wide studies when coupled with next-generation sequencing (NGS)10,11. However, these techniques require careful calibration of enzyme concentration and reaction time, along with a substantial number of cells, for successful implementation.
In 2013, the Greenleaf laboratory established the assay for transposase-accessible chromatin using sequencing (ATAC-seq) to seamlessly map chromatin accessibility at a genome-wide level using a low number of cells12. ATAC-seq takes advantage of the commercially available Tn5 transposase to preferentially insert short DNA sequences (i.e, barcodes) into open chromatin regions, typically devoid of nucleosomes, and correlates with active promoters and enhancers. The protocol requires cell permeabilization and nuclei isolation, followed by limited digestion with Tn5 for tagmentation of hyper-accessible chromatin. Later, complementary indexes are incorporated during PCR, facilitating sample multiplexing before NGS13. ATAC-seq has proven to be a robust protocol that also enables in silico transcription factor footprint and has been implemented for single-cell resolution14,15,16,17. In addition to mice and human samples, ATAC-seq has been implemented in various systems, including zebrafish and invertebrates such as Drosophila and the nematode Caenorhabditis elegans18,19,20.
For decades, C. elegans has been a powerful model system in fields such as development, cell biology, and neuroscience, leading to seminal discoveries including the identification of micro RNAs and the RNA interference (RNAi)21,22. It has a compact 100 MB genome with ~20,000 protein-coding genes23. Gene regulation occurs through promoter activity as well as distal enhancers, which have been mapped via ATAC-seq across different tissues, developmental stages, and aging time points24,18. Chromatin accessibility profiling has thus become a valuable tool for understanding gene regulation in C. elegans. Consequently, ATAC-seq holds particular promise for studying epigenetic landscapes across different environmental conditions or wild isolates to uncover diverse transcriptional responses. Additionally, it could provide insights into the chromatin architecture of other pathogenic and non-pathogenic nematodes.
One of the primary methodological challenges in C. elegans is the presence of a collagen-rich cuticle that must be dissociated to obtain a homogenous cell suspension from whole worms25. To address this challenge, we compiled and optimized different protocols to disintegrate the nematodes and then perform ATAC-seq in C. elegans18,26. Using the L4 larval stage is preferred as animals at this developmental point possess a transcriptionally active germline, which translates into open chromatin, providing a consistent and informative source of chromatin accessibility in somatic cells. Here, we present a rapid and efficient protocol for worm dissociation and cell suspension generation, enabling ATAC-seq in C. elegans. Our method is optimized for L4-stage worms and requires as little as 30 &#181;L of worm pellet, with the potential to be adapted for other developmental stages or for smaller samples, such as FACS-purified cells. This protocol can be performed in different C. elegans strains and conditions, enabling the identification of 5,000&#8211;9,000 chromatin accessibility peaks in the Bristol-N2 reference strain, laying the foundation for subsequent chromatin accessibility analyses.

<table><tbody><tr><td>250 mL, 0.22 &amp;micro;m PES</td><td>Ultra Curz</td><td>sc-200253</td><td></td></tr><tr><td>Agar</td><td>BD</td><td>214010</td><td></td></tr><tr><td>Agencourt AMPURE XP</td><td>Nalgene</td><td>A63886</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Maplica</td><td>200100</td><td></td></tr><tr><td>Calcium Chloride</td><td>Sigma</td><td>C5670-100G</td><td></td></tr><tr><td>CellTrics 20 &amp;micro;m</td><td>Sysmex</td><td>04-0042-2315</td><td></td></tr><tr><td>Cholesterol</td><td>Sigma</td><td>C8503-25G</td><td></td></tr><tr><td>Digitonin</td><td>Sigma</td><td>D141-500 mg</td><td></td></tr><tr><td>Disposable gloves</td><td>Microflex</td><td>94-243</td><td></td></tr><tr><td>DL-Dithiothreitol</td><td>sigma</td><td>D0632-10G</td><td>Harmful</td></tr><tr><td>Ethyl alcohol, pure</td><td>Sigma</td><td>1003661421</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>biowest</td><td>S181S-500</td><td></td></tr><tr><td>HEPES</td><td>Sigma</td><td>H3375-250 G</td><td></td></tr><tr><td>Illumina Tagment DNA Enzyme and Buffer (large Kit)</td><td>Illumina</td><td>20034211</td><td></td></tr><tr><td>Kapa Syber 50 mL</td><td>Sigma</td><td>KK4618</td><td></td></tr><tr><td>L-15 Medium (Leivobitz)</td><td>sigma</td><td>L4386-10L</td><td></td></tr><tr><td>Magnesium Chloride anhydrous</td><td>Sigma</td><td>M8266-100G</td><td></td></tr><tr><td>Magnesium Sulfate Heptahydrate</td><td>Sigma</td><td>M1880-500G</td><td></td></tr><tr><td>Magnesium Sulfate Heptahydrate</td><td>Sigma</td><td>M1880-500G</td><td></td></tr><tr><td>MinElute PCR Purification Kit (50 preps)</td><td>Qiagen</td><td>28004</td><td></td></tr><tr><td>NEBNext&amp;reg; High-Fidelity 2X PCR Master Mix</td><td>NEB</td><td>M0541S</td><td></td></tr><tr><td>Nonidet P40 Substitute</td><td>Sigma</td><td>74835-1L</td><td></td></tr><tr><td>Pen Strep</td><td>Gibco</td><td>15140-122</td><td></td></tr><tr><td>Peptone</td><td>BD</td><td>211677</td><td></td></tr><tr><td>Petri dish 60 mm</td><td>Tritech</td><td>T3308</td><td></td></tr><tr><td>Petri dish 90 mm</td><td>Interlux</td><td>C9015-2C</td><td></td></tr><tr><td>Pipet tips 10 &amp;micro;L, filter</td><td>Cellpro</td><td>800108</td><td></td></tr><tr><td>Pipet tips 20 &amp;micro;L, filter</td><td>Cellpro</td><td>800708</td><td></td></tr><tr><td>Pipet tips 200 &amp;micro;L, filter</td><td>Cellpro</td><td>800608</td><td></td></tr><tr><td>Potassium Chloride</td><td>Sigma</td><td>P9541 -500G</td><td></td></tr><tr><td>Potassium Phosphate Dibasic Powder</td><td>J.T. Baker</td><td>3252-01</td><td></td></tr><tr><td>Potassium Phosphate Monobasic, Crystal</td><td>J.T. Baker</td><td>3246-01 500G</td><td></td></tr><tr><td>Sodium Chloride</td><td>JT Baker</td><td>3624-01</td><td></td></tr><tr><td>Sodium dodecyl sulfate</td><td>sigma</td><td>L4509-100G</td><td></td></tr><tr><td>Sodium Hydroxide</td><td>Macron</td><td>7708-10</td><td></td></tr><tr><td>Sodium Hypochlorite</td><td>Cloralex</td><td>544394479</td><td></td></tr><tr><td>Sodium Phosphate Dibasic 7-Hydrated Crystal</td><td>J.T. Baker</td><td>3824-01</td><td></td></tr><tr><td>SPRIselect</td><td>Beckman</td><td>B23318</td><td></td></tr><tr><td>Sucrose</td><td>Sigma</td><td>S0389-500G</td><td></td></tr><tr><td>Syringe Filter PVDF 0.22 &amp;micro;m</td><td>Ultra Curz</td><td>Sc-358812</td><td></td></tr><tr><td>Trizma base</td><td>Sigma</td><td>T1503 1KG</td><td></td></tr><tr><td>Tween 20</td><td>Sigma</td><td>P9416-50ML</td><td></td></tr></tbody></table>

chromatin accessibility profiling in whole caenorhabditis elegans l4 larvae

Chromatin Accessibility Profiling in Whole Caenorhabditis elegans L4 Larvae

Chromatin accessibility plays essential roles in transcription, DNA repair, and chromosome segregation. Hyper-accessible regions usually correlate with active promoters and enhancers, facilitating transcription factor binding and regulatory activity. The assay for transposase-accessible chromatin using sequencing (ATAC-seq) enables genome-wide profiling of chromatin accessibility with very few cells. However, its implementation in Caenorhabditis elegans is limited by the nematode&#8217;s rich collagen cuticle that complicates cell dissociation. Here, we present an optimized protocol for performing ATAC-seq in whole worms at the L4 stage. The procedure begins with synchronized cultures and involves cuticle disruption, enzymatic dissociation, and cell-suspension preparation. Permeabilized nuclei are then subjected to Tn5 transposition, followed by polymerase chain reaction (PCR) amplification and purification of next-generation sequencing (NGS)&#8211;ready libraries. This protocol requires 30 &#181;L of worm pellet, can be completed in one day, and generates 5,000&#8211;9,000 accessibility peaks in the Bristol N2 reference strain. This streamlined workflow can be adapted to other developmental stages or FACS-purified cell populations. By reducing technical barriers to ATAC-seq in C. elegans, this method expands opportunities to study genome-wide chromatin accessibility in response to genetic and environmental perturbations in a whole-organism context.

Quality control after tagmentation ensures accurate library preparation 
During the protocol, it is important to pay special attention to qPCR performed after chromatin tagmentation (section 9), as it is a vital step for assessing DNA quality and the experiment’s performance. Figure 1A shows two amplification curves; the left one (under 15 cycles) represents good quality material that shows that everything was successful until this point, so it is ok to continue. The curv...

Here, we present a quick and simple ATAC-seq protocol to be implemented in L4-stage Caenorhabditis elegans whole-worm using only 30 &#181;L of worm pellet.

Chromatin accessibility plays essential roles in transcription, DNA repair, and chromosome segregation. Hyper-accessible regions usually correlate with ...

chromatin-accessibility-profiling-whole-caenorhabditis-elegans-l4

Research

JoVE Journal

Genetics

Microarray analysis was conducted to determine genetic expression profiles in C. elegans, and real-time PCR was used to validate and quantify microarray data.

Determining Genetic Expression Profiles in C. elegans Using Microarray and Real-time PCR

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Genome-wide Snapshot of Chromatin Regulators and States in Xenopus Embryos by ChIP-Seq

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Novel Metrics to Characterize Embryonic Elongation of the Nematode Caenorhabditis elegans

Here, we present a framework to relate broad-range dietary restriction to gene expression and lifespan. We describe protocols for broad-range dietary restriction and for quantitative imaging of gene expression under this paradigm. We further outline computational analyses to reveal underlying information processing features of the genetic circuits involved in food-sensing.

Quantification of Information Encoded by Gene Expression Levels During Lifespan Modulation Under Broad-range Dietary Restriction in C. elegans

We present an automated method for three-dimensional reconstruction of the Caenorhabditis elegans germline. Our method determines the number and position of each nucleus within the germline and analyses germline protein distribution and cytoskeletal structure.

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

Here, we present a combinatorial approach using high-resolution microscopy, computational tools, and single-cell labeling in living C. elegans embryos to understand single cell dynamics during neurodevelopment.

Isotropic Light-Sheet Microscopy and Automated Cell Lineage Analyses to Catalogue Caenorhabditis elegans Embryogenesis with Subcellular Resolution

This method is designed to follow formation of PRC2-mediated chromatin domains in cell lines, and the method can be adapted to many other systems.

A Method to Study de novo Formation of Chromatin Domains

This work describes a semi-high-throughput protocol that allows simultaneous 3D time-lapse imaging of embryogenesis in 80–100 C. elegans embryos in a single overnight run. Additionally, image processing and visualization tools are included to streamline data analysis. The combination of these methods with custom reporter strains enables detailed monitoring of embryogenesis.

A Semi-high-throughput Imaging Method and Data Visualization Toolkit to Analyze C. elegans Embryonic Development

A protocol for imaging the dynamic microtubules in vivo using fluorescently labeled End binding protein has been presented. We described the methods to label, image, and analyze the dynamic microtubules in the Posterior lateral microtubule (PLM) neuron of C. elegans.

In vivo Assessment of Microtubule Dynamics and Orientation in Caenorhabditis elegans Neurons

The present protocol describes a procedure for isolating intestines from adult Caenorhabditis elegans nematode worms by hand for input in genomics, proteomics, microbiome, or other assays.

Hand Dissection of Caenorhabditis elegans Intestines

This protocol describes an RNA interference and ChIP assay to study the epigenetic inheritance of RNAi-induced silencing and associated chromatin modifications in C. elegans.

Analysis of Transgenerational Epigenetic Inheritance in C. elegans Using a Fluorescent Reporter and Chromatin Immunoprecipitation (ChIP)

Learn ATAC-seq for chromatin accessibility profiling in whole Caenorhabditis elegans L4 larvae. This optimized protocol overcomes cuticle barriers and enables genome-wide epigenetic studies in one day.

Chromatin Accessibility Profiling in Whole Caenorhabditis elegans L4 Larvae

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