Chromatin Accessibility Profiling in Whole Caenorhabditis elegans L4 Larvae

Summary

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

Abstract

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’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)–ready libraries. This protocol requires 30 µL of worm pellet, can be completed in one day, and generates 5,000–9,000 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.

Introduction

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 patterns^2,3,4. Nucleosome accessibility strongly correlates with active transcription, particularly at promoters and distal regulatory regions such as enhancers, which are accessible to transcription factors

Protocol

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

1. Take a 90 mm (or two 30 mm) plate wit....

Results

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.......

Discussion

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 .......

Materials

List of materials used in this article

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