GSM8664567: Brain stem, uninfected, rep5; Mus musculus; RNA-Seq
Brain stems were harvested from animals sedated with ketamine and xylazine and thoroughly perfused with a minimum of 25 ml of PBS. Brain stems were cut into pieces and digested for 40 min at 37 °C with 5% CO2 in PBS supplemented with 1.6 IU ml-1 Collagenase/Dispase (Roche) and 0.5 mg ml-1 DNase I (Sigma-Aldrich). 20 μl of sterile EDTA was added to each sample and incubated at 37°C for another 5 min. Tissues were then mechanically disrupted through a 70um cell strainer into a single-cell suspension. Single-cell suspensions were washed with 25ml 2% FCS-HBSS (Thermo Fisher Scientific), centrifuged, and resuspended in a mixture of PBS with 0.5% BSA (VWR) and Debris Removal Solution (Miltenyi Biotec). After overlaying PBS with 0.5% BSA, suspensions were centrifuged, resuspended, and adjust the cell count to 106 cells ml-1. Single cell suspensions were converted to barcoded scRNAseq libraries following the manufacturer's instructions of Chromium Single Cell 3' Library, Gel bead & index kit and Chip G Kit (v3.1, 10x Genomics), aiming for recovering 10,000 cells per library. Quality control of libraries were performed with Tapestation 4200. After QC, libraries were sequenced on an MGI DNBSEQ-G400 sequencer.