title: GSM8714197: 6w_scRANseq_GEX; Mus musculus; RNA-Seq
library_construction_protocol: Mice were anesthetized with intraperitoneal injection of sodium pentobarbital (100 mg/kg) and perfused transcardially with PBS, each organ was removed. For the preparation of brain cells, brain parenchyma was homogenized with a potter tissue grinder in RPMI containing FBS (2% v/v), 15 mM HEPES buffer and 0.54% glucose (Modified from Masuda et al. Nat Immunol. 202037). After straining and centrifugation, the pellet was suspended in 37% Percoll (Cytiva, 17-0891-01) diluted with RPMI and cells were isolated by centrifugation at 800 × g for 20 min at 4 °C with slow acceleration and deceleration. The pellet was collected, washed with RPMI supplemented with FBS (2% v/v), and stained for flow cytometry. CD45+CD90.2+ and/or CD45+NK1.1+ cells were sorted by FACSMelody (BD Biosciences). Libraries were prepared according to the protocol of Chromuim Next GEM Single Cell 5' Reagent Kits v2 (Dual Index) (10x Genomics, PN-1000263) using a Chromium controller (10x Genomics). Briefly, GEMs were first generated by Master Mix with barcoded Single Cell VDJ 5' Gel Beads, cells and distribution to Oil to Chromium Next GEM Chip K (10x Genomics, PN-1000286). Barcoded cDNA was then generated from the GEM. This cDNA was amplified by PCR reaction to generate, 5' Gene Expression library and Feature Barcode library (10x Genomics, PN-1000190, -1000256, -1000215, and -1000250). Sequencing was then performed by Novaseq 6000 (Illumina).