NextSeq 500 sequencing; Single cell expression profiling of murine subventricular zone in adult brain three days after focal celebral ischemia
Single cell expression profiling of murine subventricular zone in adult brain three days after focal celebral ischemia
Three days after the operation, we isolated the subventricular zone and the adjacent striatum. Non-operated mice were used for a control sample (CTRL). Fluorescence-activated cell sorting was employed in order to gain CD31/45-negative and CellTrace-calcein green-positive viable cells. Focal cerebral ischemia was induced in adult males via middle cerebral artery occlusion (MCAO). Software cellranger (10X Genomics) version 3.0.2 was used to demultiplex fastq files from Illumina bcl files with default parameters. First-strand cDNA was extracted from reaction mixture during library preparation routine using Chromium next gem single-cell 3′ reagent kit (version 3.1, PN-1000128). Single-cell RNA-seq libraries were prepared using the Chromium controller instrument and Chromium next gem single-cell 3′ reagent kit (version 3.1, PN-1000128) 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 and libraries was determined using Agilent 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA).