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

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

[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. 

[STEPS]
SECTION: Tissue harversting and storage
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: Tissue harversting and storage
2. Right after dissection, tissue is fresh frozen on powdered dry ice and stored at -80 °C until processing.
SECTION: Tissue sectioning
3. Frozen hDRGs are embedded in OCT (Optimal Cutting Temperature embedding medium) in a cryomold by adding small volumes of OCT over dry ice to avoid thawing.
SECTION: Tissue sectioning
4. Tissues are cryostat sectioned at 10 μm onto SuperFrost Plus charged slides.
SECTION: Tissue sectioning
5. Slides are briefly warmed to allow the sections to adhere to the slides but are immediately returned to the −20 °C cryostat chamber until sectioning is complete.
SECTION: Tissue sectioning
6. The slides are removed from the cryostat and stored in a -80 °C freezer until shipment to AtlasXomics for further processing. Samples are shipped on dry ice.
SECTION: Transposition for Spatial ATAC-seq (AtlasXomics: AXO-0310)
7. Spatial ATAC-seq assay is performed at AtlasXomics (Product code: AXO-0310; https://www.atlasxomics.com/howitworks).
SECTION: Spatial ATAC-seq analysis (AtlasXomics: AXO-0310)
9. Processing of spatial ATAC-seq is perfomed by AtlasXomics.
SECTION: Transposition for Spatial ATAC-seq (AtlasXomics: AXO-0310)
7.1. In brief, tissue is fixed with 0.2% formaldehyde in PBS for 5 min and quenched with 1.25 M glycine for 5 min.
SECTION: Transposition for Spatial ATAC-seq (AtlasXomics: AXO-0310)
7.2. After fixation, tissue is washed with PBS and nuclease-free water and left to dry.
SECTION: Transposition for Spatial ATAC-seq (AtlasXomics: AXO-0310)
7.3. Tissue sections are permeabilized for 15 min with a buffer containing 10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.01% Tween-20, 0.1% NP40, 0.001% digitonin and 1%BSA, and washed with a solution containing 10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% Tween-20 and 1%BSA.
SECTION: Transposition for Spatial ATAC-seq (AtlasXomics: AXO-0310)
7.4. Tn5 transposition on the fixed hDRG sections is carried out at 37ºC for 30 min. Transposition reaction buffer contains tagmentation buffer, 0.1% Tween-20, 0.01% digitonin, PBS and Tn5). Transposition reaction is stopped with 40 μM EDTA for 5 min.
SECTION: Transposition for Spatial ATAC-seq (AtlasXomics: AXO-0310)
8.1. To correlate spatially barcoded accessible chromatin with tissue morphology, images of hDRG sections are acquired (Keyence BZ-X810 at 10X magnification).
SECTION: Transposition for Spatial ATAC-seq (AtlasXomics: AXO-0310)
8.2. The tissue is lysed using a reverse cross-linking solution containing 200 nM NaCl, 50 mM Tris-HCl pH 8.0, 1 mM EDTA, 1% SDS and 0.4 mg/mL proteinase K at 58°C for 2 h to release DNA fragments, which are then purified and amplified through PCR for library preparation using standard Next Generation Sequencing methods. 
SECTION: Transposition for Spatial ATAC-seq (AtlasXomics: AXO-0310)
8.4. 150X150 paired-end sequencing is performed on a NextSeq 2000 with 15% PhiX. A total of 300 million reads per sample are targeted.
SECTION: Spatial ATAC-seq analysis (AtlasXomics: AXO-0310)
9.1. Use Chromap to trim sequencing adapters, align sequenced reads to the human reference genome GRCh38/hg38 and remove duplicates from fastq files.
SECTION: Spatial ATAC-seq analysis (AtlasXomics: AXO-0310)
9.2. Fragments files containing coordinates of sequenced DNA mapping to barcodes are generated for downstream analysis.
SECTION: Spatial ATAC-seq analysis (AtlasXomics: AXO-0310)
9.3. Use AtlasXbrowser to designate pixels on tissue based on microscopy images and to create Seurat-compatible metadata files.
SECTION: Spatial ATAC-seq analysis (AtlasXomics: AXO-0310)
9.4. Use ArchR to process fragments files and remove pixels not on tissue.
SECTION: Spatial ATAC-seq analysis (AtlasXomics: AXO-0310)
9.5. At this point of the processing, the .tsv fragments file contains the filtered aligned reads which can be used to proceed with peak calling. We have provided a tissue_positions_list.csv file containing a table with rows corresponding to the 2500 spots on the array, high- and low-resolution images of the tissue, a scale_factors.json file, and experiment metadata. Together, these files can help for further processing of spatial ATAC-seq analysis using ArchR.

A brief description of some of the provided files is shown below. 

tissue_positions_list.csv
Columns on the file tissue_positions_list.csv correspond to:
Column 1: barcode
Column 2: in_tissue binary status indicating if the spot falls on a tissue spot
Column 3: array row 
Column 4: array column 
Column 5: the row pixel coordinate of the center of the spot in the full resolution image 
Column 6: the column pixel coordinate of the center of the spot in the full resolution image 

scale_factors.json
Parameters in the scale_factors.json file correspond to the relative scales of the supplied image and the array barcode features. Four aspects are provided:

fiducial_diameter_fullres: Number of pixels spanning the diameter of a fiducial spot in the original, high-resolution image
spot_diameter_fullres: Number of pixels spanning the diameter of a spot in the original, high-resolution image
tissue_highres_scalef: Scaling factor to convert pixel positions in the original, full-resolution image to pixel positions in the provided high resolution image (.png file)
tissue_lowres_scalef: Scaling factor to convert pixel positions in the original, full-resolution image to pixel positions in the provided low-resolution image (.png file)
SECTION: Transposition for Spatial ATAC-seq (AtlasXomics: AXO-0310)
8. Spatial barcoding is performed with microfluidics system (Product code: AXO-0310).
SECTION: Transposition for Spatial ATAC-seq (AtlasXomics: AXO-0310)
8.3. The PCR product is mixed with SPRI magnetic beads and incubated for 10 min. A magnetic stand is used to collect the beads, thereby purifying the final PCR product, which is eluted in nuclease-free water.