GSM6246140: Alb-vE6; Mus musculus; RNA-Seq
Mice were transcardially perfused with PBS and the cortical tissues were quickly dissected and enzymatically digested using the Neural Tissue Dissociation kit (P) (Cat#130-092-628, Myltenyi Biotec) at 37˚C in the gentleMACS™ Octo Dissociator with Heaters (Myltenyi Biotec). The myelin was then removed using magnetic bead separation (Myelin Removal Beads II, Cat#130-096-733, Myltenyi Biotec). After myelin removal and red blood cell removal (Cat.# 130-094-183, Miltenyi Biotec), microglia and debris were removed by magnetic separation using anti-CD11b+ microbeads (Cat. #130-049-601, Miltenyi Biotec), followed by a dead cell removal kit (Cat. #130-090-101, Miltenyi Biotec) according to the manufacturer's instruction. The final cell pellet was suspended in Ca/Mg-free PBS supplemented with 0.5% FBS for downstream analysis. In all samples, more than 90% of the cells were viable based on trypan blue examination. Single-cell RNA-seq (scRNAseq) was performed on the isolated cortical samples using the Chromium platform (10x Genomics, Pleasanton, CA) with the 3′ gene expression (3′ GEX) V3 kit, using a targeted input of ~ 3000 cells per sample. In brief, gel-beads in emulsions (GEMs) were generated on the sample chip in the Chromium controller. Barcoded cDNA was extracted from the GEMs using Post-GEM RT-cleanup and amplified for 12 cycles. Amplified cDNA was fragmented and subjected to end-repair, poly-A-tailing, adaptor ligation, and 10X-specific sample indexing following the manufacturer's protocol. Libraries were quantified using the TapeStation (Agilent) analysis and then sequenced on a HiSeq 4000 instrument (Illumina, San Diego, CA) targeting a depth of ~50,000 reads per cell.