GSM6355862: (Con, scRNAseq; Mus musculus; RNA-Seq
GSM6355862
Whole spinal cords were obtained from transgenic mice, and the spinal cords were stripped according to established methods and placed in ice-cold phosphate-buffered saline (PBS). The tissue was minced and dissociated with 0.25% trypsin in a centrifuge tube for 30 min at 28 °C, followed by 1 mg/mL collagenase II for 20 min, with gentle mixing every 10 min. The enzymatic reaction was terminated through the addition of 10% fetal bovine serum (FBS). The sample was filtered using a 70 µm strainer to obtain a single-cell suspension. Using single-cell 3 'Library and Gel Bead Kit V3 (10× Genomics, 1000075) and Chromium Single Cell B Chip Kit (10× Genomics, 1000074), the cell suspension (300–600 living cells per microliter determined by Count Star) was loaded onto the Chromium single cell controller (10× Genomics) to generate single-cell gel beads in the emulsion according to the manufacturer's protocol. In short, single cells were suspended in PBS containing 0.04% bovine serum albumin (BSA). Approximately 6000 cells were added to each channel, and the target cell recovered was estimated to be approximately 3000 cells. Captured cells were lysed, and the released RNAs were barcoded through reverse transcription in individual GEMs. Reverse transcription was performed on an S1000TM Touch Thermal Cycler (Bio Rad) at 53 °C for 45 min followed by 85 °C for 5 min, and incubated at 4 °C. The complementary DNA (cDNA) was generated and then amplified, and the quality was assessed using an Agilent 4200. According to the manufacturer's instruction, scRNA-seq libraries were constructed using Single Cell 3' Library and Gel Bead Kit V3. The libraries were finally sequenced using an Illumina Novaseq6000 sequencer with a sequencing depth of at least 100,000 reads per cell with a pair-end 150 bp reading strategy.