GSM9112625: brain cells, Sham; Mus musculus; RNA-Seq
In brief, injured cortical tissues were minced and enzymatically dissociated using 8U/ml papain and 125 U/ml DNase I. Tissues were mechanically dissociated by gentle pipetting until no visible chunks were present and then terminated by adding 10% ovomucoid. Cell clusters were removed by filtering through 70μm strainers and debris were discarded using 30% Percoll density gradient centrifugation in choline chloride solution. Cell viability was measured by Cellometer Auto 2000 Automatic Cell Viability Counter system (Nexcelom, USA) and samples with > 85% viability were proceeded to scRNA-seq library preparation. Approximately 5,188~11,934 cells for each sample were loaded according to the instructions of 10X Chromium single-cell platform (Supplemental Figure S1b). The libraries were then purified, quantified and sequenced on an Illumina HiSeq (Illumina, San Diego, CA, USA). Single-cell suspensions were processed for library preparation using the 10x Genomics Chromium Single Cell 3' Reagent Kits v3 according to the manufacturer's instructions (10x Genomics, Pleasanton, CA, USA). Briefly, viable single cells were encapsulated with barcoded gel beads and reverse transcription reagents in nanoliter-scale Gel Bead-In-Emulsions (GEMs) using the Chromium Controller. Within each GEM, cell lysis and barcoded reverse transcription of RNA occurred to generate uniquely labeled cDNA for each cell. After reverse transcription, GEMs were broken, and cDNA was recovered and amplified by PCR. Amplified cDNA was subjected to enzymatic fragmentation, end repair, A-tailing, and adaptor ligation. Fragmented cDNA was then amplified with sample index primers to generate sequencing-ready libraries.  Library quality and concentration were assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) and Qubit Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA). Libraries were pooled and sequenced on the Illumina HiSeq platform with a paired-end 150 bp configuration to achieve sufficient depth for transcriptome coverage.