title: GSM8415530: naive, 3 mice pooled, scRNAseq GEX; Mus musculus; RNA-Seq
library_construction_protocol: Mice were perfused with ice-cold saline and brains were gently removed. The hemispheres were split, the white matter and meninges were removed with forceps. The tissue was dissociated using the papain dissociation system (#LK003150, Worthington Biochemical Corporation) according to the manufacturer's protocol and an incubation time of 15min. The cell suspension was cleared from dead cells and cell debris using the dead cell removing kit (#130-090-101, Miltenyi). Propidium iodide negative live single cells were flow cytometrically sorted using a BD Fusion cell sorter into RPMI medium containing 5% fetal bovine serum. The cell suspension was centrifuged and washed with PBS containing 0.04% bovine serum albumin (BSA). The cells were resuspended in PBS containing 0.04% BSA for a concentration of 1,200cells/µl, counted and loaded into the Chromium Next GEM Chip G (PN-1000127, 10x Genomics) for a target recovery of 10,000 cells according to the 10x Genomics Next GEM Single Cell 3' v3.1 (Dual Index) kit protocol (Step 1, CG000315, Rev E) for GEM preparation (PN-1000296, 10x Genomics) and run in a 10x Genomics Chromium Controller. After GEM preparation, the protocol of the kit was followed for post GEM-RT Cleanup & cDNA amplification (Step 2) and 3'gene expression library construction (Step 3). Single-cell libraries were sequenced using an Illumina HiSeq 1500 sequencer with a sequencing depth of >20,000 reads per cell. Libraries were performed according to the manufacter's instructions (single cell 3' v3.1 protocol, 10x Genomics).