Aug 20, 2024
Human Dorsal Root Ganglion bulk ATAC-seq protocol
- Úrzula Franco-Enzástiga1,
- Theodore Price1
- 1University of Texas at Dallas
- PRECISION Human Pain Network
Protocol Citation: Úrzula Franco-Enzástiga, Theodore Price 2024. Human Dorsal Root Ganglion bulk ATAC-seq protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvokx27l4o/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: March 21, 2024
Last Modified: August 20, 2024
Protocol Integer ID: 97116
Keywords: ATAC-seq, bulk ATAC-seq, chromatin, epigenomics, human DRG, seq on fresh human dorsal root ganglia, fresh human dorsal root ganglia, bulk atac, seq protocol, seq protocol in this protocol, seq
Funders Acknowledgements:
National Institutes of Health
Grant ID: U19NS130608
National Institutes of Health
Grant ID: R01NS111929
Abstract
In this protocol, we describe how to perform bulk ATAC-seq on fresh human dorsal root ganglia.
Materials
Key materials
Buffers:
Ice-cold bubbled NMDG-aCSF pH 7.4
- 93 mM NMDG
- 2.5 mM KCl
- 1.25 mM NaH2PO4
- 30 mM NaHCO3
- 20 mM HEPES
- 25 mM glucose
- 5 mM ascorbic acid
- 2 mM thiourea
- 3 mM sodium pyruvate
- 10 mM Mg2SO4
- 0.5 mM CaCl2
- 12 mM N-acetylcysteine; osmolarity 310 mOsm
Ice-cold nuclei isolation buffer
- 250 mM sucrose
- 25 mM KCl
- 5 mM MgCl2
- 10 mM Tris-HCl pH 8.0
- 0.1% Triton X-100 - Sigma-Aldrich (Cat. No. T8787-50mL)
Materials and equipment required:
- ATAC-seq kit - Active Motif kit (Cat. No. 53150)
- DKW tube for KIMBLE® KONTES® Potter-Elvehjem Tissue Dounce homogenizer, Size 19, 1 mL (Cat. No. 885752-0019) with PTFE pestle
- Sterile 40-μm cell strainer - CELLTREAT (Cat. No. 229481)
- Costar® 2 mL tube - Corning (Cat. No. 3213)
- Ultrapure DNase/RNase Free Distilled Water - Thermo Scientific (Cat. No. 10977015)
- Centrifuge 5702R - Eppendorf® (Cat. No. 05-414-126)
- Magnetic separator - 10X (Cat. No. 120250)
- Trypan blue - Gibco (Cat. No. 15-250-061)
- ThermoMixer C - Eppendorf® for 1.5 mL tubes (Cat. No. EP5382000023)
- NextSeq 2000 sequencer
- Bonn scissors - Fine Science Tools (Cat. No. 14184-09)
- Dumont #5 Forceps - Fine Science Tools (Cat. No. 11252-50)
- Microcentrifuge
- Hematocytometer
- 100% ethanol
- Wet ice
- Ice bucket
- 5 cm petri dish
- Pipettes and corresponding pipette tips
Troubleshooting
Before start
Required PPE: All work must be done wearing appropriate PPE including lab coat and gloves. Proper safety precautions should be taken into account while working with and disposing human tissue. The use of sterile tools and proper cleaning and sterilization before and after every use is recommended.
Tissue harvesting and cleaning procedure
Surgically excised lumbar L4 or L5 human dorsal root ganglia (hDRGs) are obtained from organ donors at Southwest Transplant Alliance (STA) at 2 h post cross-clamp.
Right after dissection, hDRGs are transported in cold NMDG-aCSF pH 7.4 (see solution components in Materials) bubbled with carbogen gas (95% O2, 5% CO2) in wet ice to the laboratory.
A petri dish containing ice-cold NMDG-aCSF pH 7.4 is used to place the hDRG, ensuring the petri dish remains on ice throughout the cleaning process.
Bonn scissors are used to remove fat and connective tissue surrounding the DRG. Forceps are used to remove dura by pulling it away from the hDRG.
Nuclei isolation
Cleaned hDRG is placed in an eppendorf tube containing 2 mL of ice-cold nuclei isolation buffer (see solution components in Materials).
hDRG is chopped into small pieces using scissors.
The total volume is transferred to a Dounce homogenizer. Homogenization requires 5 strokes of the loose pestle and 15 strokes of the tight pestle applied to the tissue.
The homogenate is transferred to a conical tube (2 mL) and centrifuged at 100 x g for 8 min at 4 °C.
The supernatant is carefully removed without disrupting the soft pellet.
The pellet is resuspended in 2 mL of triton-free nuclei isolation buffer.
The resuspended pellet is centrifuged at 100 x g for 8 min at 4 °C.
The supernatant is removed without disrupting the pellet. At this step most of the pellet are nuclei.
Nuclei are resuspended in 2 mL of nuclei isolation buffer without triton and filtered through a 40-μm cell strainer.
Nuclei counting
The number of isolated nuclei is counted using a hematocytometer with 0.4% trypan-blue (1:1 ratio) to stain the nuclei.
A total of 100,000 nuclei are aliquoted and centrifuged at 500 x g for 10 min at 4 °C. The supernatant is removed to proceed to tagmentation.
Tagmentation
Tagmentation is performed by following the instructions provided in the Active Motif kit (https://www.activemotif.com/documents/2182.pdf) (Cat. No. 53150) from the Tagmentation Reaction and Purification section until the end of the protocol. 50 μL of Tagmentation Master Mix (Page 5 of the protocol), prepared right before starting the tagmentation step, are added to each sample.
The tagmentation reaction is incubated at 37 °C for 30 min in a thermomixer at 800 rpm.
Tagmented DNA purification
250 μL of purification binding buffer and 5 μL of 3 M sodium acetate are added.
DNA purification columns are employed to isolate the DNA.
Tagmented DNA is eluted in 35 μL of DNA elution buffer.
Library preparation
The amplification of tagmented DNA through PCR is carried out using i7-and i5-Illumina's Nextera-based adapters as per the provider's instructions (Cat. No. 53150).
ATAC sequencing
Pair-end 75 cycle sequencing reads are acquired on the NextSeq 2000 sequencer. A total of 100 million reads are intended per sample.
Processing of ATAC-seq data
Raw bulk ATAC-seq data undergo a process of pre-alignment quality control, read alignment to the reference genome, and post-alignment processing as described below.
Use FastQ to verify the initial quality of raw sequencing using fastq files.
Use TrimGalore to remove adapters and verify quality of trimming using FastQ.
Use Bowtie2 to map paired-end reads to the reference genome GRCh38/hg38.
Use Samtools to sort and filter reads by removing unmapped and low quality reads, and Bamtools to remove mitochondrial DNA reads.
Remove duplicates using Picard.
At this point of the processing the .bam file contains the filtered aligned reads which can be used to proceed with peak calling.
Protocol references
Buenrostro JD, Wu B, Chang HY, & Greenleaf WJ (2015) ATAC-seq: A Method for Assaying Chromatin Accessibility Genome-Wide. Curr Protoc Mol Biol 109:21 29 21-21 29 29.
Valtcheva MV, et al. (2016) Surgical extraction of human dorsal root ganglia from organ donors and
preparation of primary sensory neuron cultures. Nat Protoc 11(10):1877-1888.
Mich JK, et al. (2021) Functional enhancer elements drive subclass-selective expression from
mouse to primate neocortex. Cell Rep 34(13):108754.