single-cell RNA-seq for specimen F02, sample GW002, isolated Microglia ex vivo
Sorted microglia were centrifuged for 5 minutes at 300 rcf and the supernatant was carefully aspirated leaving approximately 25 μl behind. Cells were resuspended in up to 40 μl reaction buffer (0.1% BSA (SRE0036-250ML, Sigma) and 1 U/μl RNasin inhibitor (PAN21110, Promega) in PBS. 12,000 cells (viability 65-100%) were loaded onto a Chromium Controller (10x Genomics). Libraries were generated according to manufacturer specifications (Chromium Single Cell 3' Library and Gel Bead Kit v3, 1000075; Chromium Single Cell 3' Library Construction Kit v3, 100078; Chromium Chip B Single Cell Kit, 1000153; Single Index Kit T Set A, 1000213). cDNA was amplified for 12 PCR cycles. SPRISelect reagent (Beckman Coulter) was used for size selection and clean-up steps. Final library concentration was assessed by Qubit dsDNA HS Assay Kit (Thermo-Fischer Scientific) and fragment size was checked using the High Sensitivity D1000 ScreenTape assay on a Tapestation 4200 (Agilent) to ensure that fragment sizes were distributed normally about 500 bp.