TITLE: Human Dorsal Root Ganglion bulk ATAC-seq protocol
AUTHORS: Úrzula Franco-Enzástiga, Theodore Price

[DESCRIPTION]
In this protocol, we describe how to perform bulk ATAC-seq on fresh human dorsal root ganglia. 

[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.

[STEPS]
SECTION: Tissue harvesting and cleaning procedure
1. 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. 
SECTION: Nuclei isolation
5. Cleaned hDRG is placed in an eppendorf tube containing 2 mL of ice-cold nuclei isolation buffer (see solution components in Materials). 
2. 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. 
4. 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.
3. 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.
SECTION: Nuclei isolation
6. Nuclei are resuspended in 2 mL of nuclei isolation buffer without triton and filtered through a 40-μm cell strainer.
SECTION: Nuclei counting
7. The number of isolated nuclei is counted using a hematocytometer with 0.4% trypan-blue (1:1 ratio) to stain the nuclei. 
SECTION: Nuclei counting
8. 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.
SECTION: Tagmentation
9. 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. 
SECTION: Tagmentation
10. The tagmentation reaction is incubated at 37 °C for 30 min in a thermomixer at 800 rpm.
SECTION: Tagmented DNA purification
11. 250 μL of purification binding buffer and 5 μL of 3 M sodium acetate are added.
SECTION: Tagmented DNA purification
12. DNA purification columns are employed to isolate the DNA.
SECTION: Tagmented DNA purification
13. Tagmented DNA is eluted in 35 μL of DNA elution buffer.
SECTION: Library preparation
14. 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).
SECTION: ATAC sequencing
15. Pair-end 75 cycle sequencing reads are acquired on the NextSeq 2000 sequencer. A total of 100 million reads are intended per sample.  
SECTION: Processing of ATAC-seq data
16. 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.
SECTION: Processing of ATAC-seq data
17. At this point of the processing the .bam file contains the filtered aligned reads which can be used to proceed with peak calling. 
SECTION: Nuclei isolation
5.1. hDRG is chopped into small pieces using scissors.
SECTION: Nuclei isolation
5.2. 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.
SECTION: Nuclei isolation
5.3. The homogenate is transferred to a conical tube (2 mL) and centrifuged at 100 x g for 8 min at 4 °C. 
SECTION: Nuclei isolation
5.4. The supernatant is carefully removed without disrupting the soft pellet. 
SECTION: Nuclei isolation
5.5. The pellet is resuspended in 2 mL of triton-free nuclei isolation buffer.
SECTION: Nuclei isolation
5.6. The resuspended pellet is centrifuged at 100 x g for 8 min at 4 °C.
SECTION: Nuclei isolation
5.7. The supernatant is removed without disrupting the pellet. At this step most of the pellet are nuclei.
SECTION: Processing of ATAC-seq data
16.1. Use FastQ to verify the initial quality of raw sequencing using fastq files.
SECTION: Processing of ATAC-seq data
16.2. Use TrimGalore to remove adapters and verify quality of trimming using FastQ.
SECTION: Processing of ATAC-seq data
16.3. Use Bowtie2 to map paired-end reads to the reference genome GRCh38/hg38.
SECTION: Processing of ATAC-seq data
16.4. Use Samtools to sort and filter reads by removing unmapped and low quality reads, and Bamtools to remove mitochondrial DNA reads.
SECTION: Processing of ATAC-seq data
16.5. Remove duplicates using Picard.