GSM6126034: SCI1M, scRNAseq; Mus musculus; RNA-Seq
One-centimeter segment encompassing the epicenter of the injury or matched non-injured area was harvested for tissue dissociation (including 18 cords in Naïve, 9 cords in Sham, and 8 cords in SCI1M group). After the mice were euthanized, transcardial perfusion with cold PBS was performed and cords were dissociated using a mixed digestion solution of papain, trypsin-EDTA, DNase I and L-cysteine in DMEM supplemented with BSA at 37°C under gentle shaking for 15 mins. After digestion solution was removed by centrifugation, cells were triturated with fire-polished glass Pasteur pipettes in fresh DMEM supplemented with 0.5% BSA. The supernatant containing cells were separated from the undissociated tissue at the bottom and stored at 4°C. The undissociated tissue was processed through above steps once more. Cell suspension were collected, passed through 70-µm strainer, and purified by density gradient centrifugation with 4% BSA-supplemented DMEM to eliminate remaining tissue clots and dead cells. To remove myelin, cells were then resuspended in a cold DPBS supplemented with 0.5% BSA containing Myelin Removal Beads II (Miltenyi Biotec; cat #130-096-733) following the manufacturer's protocol. Next, tdT+/DAPI- cells for scRNA library preparation were collected by FACS using a BD FACS Aria II flow cytometer (BD Biosciences) with a 100 µm nozzle at 4°C. Samples were then washed in DMEM with 10% fetal bovine serum to reach the concentration at 400-1000 cells per ul. Cells from each sample were stained with Trypan Blue and cell viability was calculated on hemocytometer before loading on 10x Chromium. Naïve, Sham and SCI1M samples were sequenced. Sample libraries were prepared by The University of Houston Sequencing and Editing Core (UH-SNEC) based on Chromium Single Cell 3' Library and Gel Bead Kit instructions (v3-v3.1). All samples were indexed with Single Index Kit T Set A (10X Genomics; PN-1000213) or Chromium i7 Multiplex Kits (10X Genomics; PN-120262). Paired-end sequencing was performed using the NovaSeq 6000 (Sham and SCI1M) or NextSeq 500 (Naïve) sequencing instrument at recommended settings (R1: 28 cycles; i7 index: 8 cycles; i5 index: none; and R2: 91 cycles).