title: GSM9012052: Sciatic nerve myeloid cells, Crush surgery, 15 weeks after surgery, CSP; Mus musculus; RNA-Seq
library_construction_protocol: 1 week or 15 weeks after surgery, mice were transcardially perfused with 10 ml PBS to remove leukocytes from circulating blood. Ligated, crushed or uninjured (sham) nerves were dissected and placed into a petri dish containing F12 medium (Gibco, #21765-029). Ligatures were removed with tweezers, if still present, and nerves were cut to 0.5 cm, ensuring equal lengths of nerve either side of the ligation/crush site. Following dissections, samples were transferred into wells of a standard 96-well PCR plate containing 51μl of the following digestion mix: 6.25 mg/ml collagenase type IA (Sigma Aldrich, #C9891), 0.2% Pronase (Millipore, #53702), 0.4% Hyaluronidase (ABNOVA, #P52330) in F12 medium. Spring scissors were used to chop the tissue into small pieces (50x chops, cleaning scissors between samples with 100% ethanol). After this manual dissociation, the plate was incubated on a shaking platform (220 rotations per minute) at 37°C for 30 minutes. The digestion mix was subsequently removed and replaced with 100μl of fluorescence-activated cell sorting (FACS) buffer: 0.4% BSA (Sigma-Aldrich, #A3983), 15 mM HEPES (Gibco, #15630080), 2mM EDTA (Invitrogen, #15575038) in Hank's Buffered Saline Solution (HBSS, diluted from 10x, Gibco, #14175095). Samples were then further dissociated by pipetting up and down 50x, with the resulting single-cell solution filtered through a 35μm cap of a BD falcon tube with a cell strainer cap (BDBiosciences, # 352235). The plate was centrifuged at 1500 rotations per minute for 5 minutes at 4°C, after which supernatants were removed and resuspended in 50μl of the following staining mixture for 30 minutes at 4°C in a dark fridge: Fc block (BioLegend, #101302, 1:200), FITC-CD45 to stain leukocytes (BioLegend, #147709, 1:1200), PE-Cy7-CD11b to stain myeloid lineage cells (BioLegend, #101215, 1:1200) and, depending on the sample, one of four hashtag antibodies: TotalSeq-B0301-MHCI (BioLegend, #155831), TotalSeq-B0302-MHCI (BioLegend, #155833), TotalSeq-B0303-MHCI (BioLegend, #155835), TotalSeq-B0304-MHCI (BioLegend, #155837). At the acute time point, one week after surgery, the antibody staining mix also contained BUV395-Ly6G to stain neutrophils (BD Bioscience, #563978, 1:300). After staining, cells were centrifuged for 5 minutes at 1,500 rotations per minute at 4°C, after which the cell pellets were resuspended in 300μl FACS buffer and transferred to FACS tubes, adding DAPI to stain for dead cells (Invitrogen, #D1306, 1μl of a 1:2 dilution). Sorting was performed on a BD FACSAria II or BD FACSAria Fusion in the Advanced Cytometry Platform Core Facility of the Guy's and St Thomas' NHS Foundation Trust. Unstained cells, DAPI single stained cells and single stained control beads (BD™ CompBeads, BD Bioscience, #552845) were used for compensation. Cells were sorted into 1.5ml microcentrifuge tubes containing 4μl of 4% BSA (Sigma-Aldrich, #A3983) in PBS. All tubes were pre-coated for at least 1 hour with the same 4% BSA solution and placed on ice immediately following cell collection. The following samples were collected: for the 1-week time point, CD45+/CD11b+/Ly6G- live single cells from n = 2 sham (2,000 cells each), crush and PSNL mice (8,000 cells each); for the 15-week time point, as many CD45+/CD11b+ live single cells as possible were collected from n = 3 crush and n = 4 PSNL mice. Hashed samples were combined as follows in two separate 10X runs: for the 1-week time point, one 10X well (acute crush) was filled with cells from 1x male sham, 1x male crush, 1x female crush mouse, while the other (acute PSNL) was filled with cells from 1x female sham, 1 male PSNL and 1 female PSNL mouse. A separate 10X run was performed for the 15-week time point, with one well (chronic crush) filled with 2x female crush and 1x male crush sample, and the other well (chronic PSNL) filled with 2x female and 2x male PSNL samples. Libraries were generated using the 10X Genomics Chromium Single Cell 3ʹ Feature Barcode Library & Gel Bead Kit (v3 Chemistry) according to manufacturer's instructions and sequenced on an Illumina NextSeq 2000 P2 Flow Cell obtaining mean reads per cell of: 24,752 (acute crush well), 35,710 (acute PSNL well), 45,531 (chronic crush well), and 59,220 (chronic PSNL well).