GSM8009063: EAE_SC Tag; Mus musculus; RNA-Seq
Twenty-three days after induction of EAE or stroke, mice were anesthetized by inhalation with isoflurane, and after PBS perfusion, the brain parenchyma was removed and homogenized in 2% FBS/RPMI solution containing glucose and HEPES. After straining and centrifugation at 400 x g for 5 minutes, the pellet was resuspended in 37% Percoll (Cytiva), cells were isolated by centrifugation with slow acceleration/deceleration 800 x g for 20 minutes and resuspended with filtered 0.5% BSA/PBS. Totalseq-Hashtag antibody (BioLegend) was incubated with each sample, and CD45.2+ cells were sorted by FACSMelody (BD Biosciences). Libraries were prepared according to the protocol of the Chromuim Next GEM Single Cell 5' Reagent Kits v2 (Dual Index, 10x Genomics, Pleasanton, CA, USA) using a Chromium controller (10x Genomics). Briefly, GEMs were prepared by loading barcoded Single Cell VDJ 5' Gel Beads, master mix containing cells and RT enzymes, and Oil onto Chromium Next GEM Chip K. Barcoded cDNA was then generated from the GEM. This cDNA was amplified by PCR reaction to generate V(D)J, 5' Gene Expression and Feature Barcode libraries.