GSM8968449: brain cells, FACS sorted, 4month old, ctrl, rep1; Mus musculus; RNA-Seq
Mice were anesthetized using ketamine/xylazine and perfused with cold PBS. One hemisphere including cortex and hippocampus but excluding cerebellum and brain stem were dissociated with a previously established protocol using gentleMACS OctoDissector together with the Papain Neural Tissue Dissociation Kit (Miltenyi Biotec) and actinomycin D (catalog no. A1410, Sigma-Aldrich). After dissociation, myelin debris were removed from the single cell suspension using Myelin Removal Beads II (Miltenyi Biotec). The single cells were then resuspended in 0.04% BSA + PBS and counted using an automatic cell counter and trypan blue (BioRad TC20). For generating the sorted scRNA-seq data, 16 weeks and 32 weeks old Plp1Tg mice were anesthetized using ketamine/xylazine and perfused with cold PBS. Following gentleMACS brain hemisphere dissociation, SYTOX Blue-negative cells were sorted into a 2-ml Eppendorf tube with 1 ml of PBS + 5% fetal bovine serum (FBS). Sorted cells were centrifuged at 300g for 10 min at 4 °C. Cell pellets were resuspended in 0.04% bovine serum albumin (BSA) + PBS catching medium at a concentration of 700–900 cells per µl. Single cell libraries were constructed according to the 10X Genomics instructions (Chromium Single Cell 3' v3.1 (Single Index). Sample processing and library preparation were performed according to the manufacturer's instructions using AMPure beads (Beckman-Coulter). Sequencing was performed on the Illumina NovaSeq 6000 (BGI Group).