title: NextSeq 500 paired end sequencing; Single cell expression profiling of NG2 cell lineage from adult mice cortex three days after focal cerebral ischemia
design: Single cell expression profiling of NG2 cell lineage from adult mice cortex three days after focal cerebral ischemia
library_construction_protocol: Three days after surgery, we isolated the cerebral cortex adjacent to the site of injury. Fluorescence-activated cell sorting was used to obtain CD31/45 negative and CellTrace-calcein green positive viable cells carrying tdTomato/NG2 red fluorescence. In adult males, middle cerebral artery occlusion (MCAO) induced focal cerebral ischemia. For healthy tissue (CTRL), sham-operated mice were used.  Software cellranger (10X Genomics) version 6.1.1 was used to demultiplex fastq files from Illumina bcl files with default parameters. First-strand cDNA was extracted from cell suspension (8 individuals pooled per group) during library preparation routine using Chromium next gem single-cell 3′ reagent kit (version 3.1). Single-cell RNA-seq libraries were prepared using the Chromium controller instrument and Chromium next gem single-cell 3′ reagent kit (version 3.1) according to the manufacturer's protocol (both 10× Genomics, Pleasanton, CA, USA) targeting at 4000 cells per sample. The quality and quantity of the resulting cDNA libraries was determined using Agilent 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA).