GSM8412893: WT_Sham1; Mus musculus; RNA-Seq
Brain tissue was enzymatically digested using collagenase 1 and mechanically ground to obtain a single-cell suspension. The cell suspension was filtered through a 70-micron cell strainer to remove large tissue fragments. The cell suspension was centrifuged (400 g, 10 minutes) to precipitate the cells. The supernatant was removed, and the cells were resuspended in cell separation buffer and repeated 2-3 times. According to the instructions of the magnetic bead supplier, 10 microliters of Cd11b magnetic beads were added to the resuspended cells and incubated at 4°C for 20-30 minutes. The labeled cells were washed with cell separation buffer to remove unbound magnetic beads. The cell suspension was sorted through a magnetic separation column. Cd11b-positive cells were eluted using cell separation buffer. Finally, the sorted Cd11b-positive cells were subjected to single-cell sequencing. Cell numbers and viabilities were validated by Cellometer prior to single cell RNAseq library preparation. For 10x single cell RNAseq assay,  qualified cells from the sham, TBI, and TBI+4-OI groups, respectively were targeted by using 10x Single Cell 3' RNAseq technology provided by 10x Genomics (10X Genomics Inc, CA). Briefly, viable single cell suspensions were partitioned into nanoliterscale Gel Beads-In-EMulsion (GEMs). mRNA was extracted from single cell suspension using the 10X Genomics Chromium™ Single Cell G Chip Kit. Library construction was performed according to 10x Genomics Chromium™ Single Cell 3' Reagent Kit v3.1 protocol. Following enzymatic fragmentation, end-repair, A-tailing, and adaptor ligation, single cell 3' libraries comprising standard Illumina P5 and P7 paired-end constructs were generated. Library quality controls were performed by using Agilent High Sensitive DNA kit with Agilent 2100 Bioanalyzer and quantified by Qubit 2.0 fluorometer. Pooled libraries at a final concentration of 750pM were sequenced with paired end single index configuration by Illumina NextSeq 2000.