TITLE: Single-cell RNA sequencing from human dorsal root ganglion
AUTHORS: Ishwarya Sankaranarayanan, Juliet M Mwirigi, Joesph B Lesnak, Muhammad Saad Yousuf, Theodore Price

[DESCRIPTION]
This protocol describes how to extract single cells from dorsal root ganglia sourced from human organ donors and perform single-cell RNA sequencing using the Single Cell Gene Expression FLEX kit from 10X Genomics.

[BEFORE_START]
Required PPE: Ensure proper personal protective equipment (PPE), including a lab coat and gloves, is always worn. Before beginning,  thoroughly clean pipettes and the lab bench with 70% ethanol, followed by an RNase decontamination solution like RNaseZap

[STEPS]
SECTION: Harvesting and isolating single cell from human dorsal root ganglia
1.4. The extracted DRGs are transferred to a sterile petri dish on ice, where they are carefully trimmed using forceps and Bonn scissors to remove connective tissue, fat, spinal, and peripheral nerve processes. The dural layers (perineurium and epineurium) are meticulously removed, leaving only the ganglia bodies.
SECTION: Harvesting and isolating single cell from human dorsal root ganglia
1.1.  The human dorsal root ganglion immediately placed in chilled and bubbled aCSF, containing:
93 mM N-Methyl-d-glucamine (NMDG),
HCl (12 N): to adjust pH
2.5 mM KCl,
1.25 mM NaH2PO4,
30 mM NaHCO3,
20 mM HEPES,
25 mM d-(+)-Glucose,
5 mM l-Ascorbic acid,
2 mM Thiourea,
3 mM Sodium pyruvate,
10 mM MgSO4, 
0.5 mM CaCl2 dihydrate,
12 mM N-acetylcysteine. 
The solution was made to pH 7.4.

SECTION: Library preparation for single-cell RNA sequencing
3. The cells are then incubated for 16 hours with the probe hybridization provided by 10X Genomics (Chromium Fixed RNA profiling kit, Human Transcriptome, 1000476).
SECTION: Processing of single-cell RNA sequencing
4. Sequencing data were processed and mapped to the human (GRCh38) genome using 10X Genomics Fix RNA profiling Cellranger multi pipeline (v7) 
SECTION: Processing of single-cell RNA sequencing
4.1. The processed sequencing data output from the Cellranger pipeline can be further analyzed using packages such as Seurat or Scanpy. 
SECTION: Harvesting and isolating single cell from human dorsal root ganglia
1. Human dorsal root ganglia (DRGs) from lumbar levels L3-L5 are surgically extracted from organ donors with no known history of chronic pain at the Southwest Transplant Alliance (STA) 2-4 hours after cross-clamp. 
SECTION: Harvesting and isolating single cell from human dorsal root ganglia
1.2. The DRGs were transported from the tissue recovery site to the University of Texas at Dallas for further isolation.
SECTION: Harvesting and isolating single cell from human dorsal root ganglia
1.5. These ganglia are further divided into approximately 2 mm thick sections using Bonn scissors.
SECTION: Harvesting and isolating single cell from human dorsal root ganglia
1.6. The tissue fragments are placed in sterile 5 mL of prewarmed enzyme mix containing Stemxyme (2mg/ml) and Deoxyribonuclease I (0.1mg/ml) in sterile filtered Hank's Balanced Salt Solution (HBSS) without calcium and magnesium.
SECTION: Harvesting and isolating single cell from human dorsal root ganglia
1.7. This mixture is then subjected to enzymatic digestion in a shaking water bath at 37◦C. The DRGs are gently triturated through a sterile fire-polished glass Pasteur pipette every 25 minutes until the solution becomes cloudy and the tissue chunks smoothly pass through the pipette without resistance. This process takes roughly three to four hours.
SECTION: Harvesting and isolating single cell from human dorsal root ganglia
1.8. Following enzymatic digestion, the dissociated DRGs are passed through a 100 µm cell strainer to remove debris and obtain a uniform cell suspension.
SECTION: Harvesting and isolating single cell from human dorsal root ganglia
1.9. The DRG cells are further purified by layering the cell suspension on a 3ml of 10% Bovine Serum Albumin (BSA) solution prepared in sterile HBSS.
SECTION: Harvesting and isolating single cell from human dorsal root ganglia
1.10. The BSA gradient is then centrifuged at 900g for 5 minutes at room temperature, resulting in a cell pellet of DRG cells.
SECTION: Harvesting and isolating single cell from human dorsal root ganglia
1.11. The supernatant was removed, and the cell pellet was washed and resuspended in 1 ml of PBS. A 10 μl aliquot of the cell suspension was mixed with an equal volume of 0.4% Trypan blue and used for counting with a hemocytometer to calculate the total number of cells accurately.
SECTION: Library preparation for single-cell RNA sequencing
3.3. The libraries were sequenced using Nextseq2000 at the University of Texas at Dallas Genome Core.
SECTION: Harvesting and isolating single cell from human dorsal root ganglia
1.12. The cells were centrifuged at 850g for 5 minutes, and the cell pellet is ready for fixation.
SECTION: Fixation and probe hybridization for single-cell RNA sequencing
2.4. The fixed samples can be stored for one week at 4ºC (short-term storage) or six months at -80ºC (long-term storage) by adding 50% glycerol for a final concentration of 10% before proceeding to the library preparation.
SECTION: Fixation and probe hybridization for single-cell RNA sequencing
2.3. The supernatant is removed, and the cell pellet is resuspended in 1 ml of quenching buffer containing 100µl of enhancer, as provided in the 10X single-cell fixed RNA sample preparation kit (1000414)
SECTION: Fixation and probe hybridization for single-cell RNA sequencing
2.2. The fixed cells are resuspended in 2 ml of phosphate buffer saline (PBS) and centrifuged at 850g for 5 minutes.
SECTION: Fixation and probe hybridization for single-cell RNA sequencing
2.1. The whole cells are then fixed for 17 hours at 4ºC.
SECTION: Fixation and probe hybridization for single-cell RNA sequencing
2. The cell pellet is immediately fixed using the Chromium Next GEM Single Cell Fixed RNA Sample Preparation Kit.  300,000- 10X106 cells per one ml of the fixation buffer is added to the cell pellet and carefully pipetted up and down to create a homogeneous cell suspension.
https://cdn.10xgenomics.com/image/upload/v1704391365/support-documents/CG000478_DemonstratedProtocol_Cell_NucleiFixation_Chromium_FixedRNA_Profiling_RevD.pdf
SECTION: Library preparation for single-cell RNA sequencing
3.1. NOTE: To compare other datasets, it is essential to maintain the same fixation and
probe hybridization times for subsequent single-cell sequencing experiments.
SECTION: Library preparation for single-cell RNA sequencing
3.2. The library preparation is completed according to the manufacturer’s protocol.
https://cdn.10xgenomics.com/image/upload/v1722287750/support-documents/CG000527_Chromium_FixedRNAProfiling_MultiplexedSamples_UserGuide_Rev_F.pdf)
SECTION: Harvesting and isolating single cell from human dorsal root ganglia
1.3. A detailed protocol on tissue recovery, preparation of aCSF, and transport can be found at dx.doi.org/10.17504/protocols.io.kqdg32qr1v25/v1