GSM3484669: Adult2; Mus musculus; RNA-Seq
To acquire lung tissue single cell suspensions, tissue was minced and fragments were digested in 5 ml DMEM containing collagenase IV (142.5 U/ml; Thermo Fisher, 17104019) and DNase I (118,050 U/ml, Sigma-Aldrich, 9003-98-9) for 25 minutes at 37°C on a shaker (150 rpm). The single cell suspension was filtered through a 70μm cell strainer and washed once with 5 ml DMEM containing 10% FBS and Penicillin-Streptomycin (10,000 U/mL). The acquired cells were enriched for leukocytes by a 36% Percoll (Sigma-Aldrich, P4937) gradient media and red blood cell lysis.   Adult or neonatal (12-day-old) RORα-YFP mouse lungs were process as described. Pooled adult or neonatal single cell suspensions were incubated with anti-mouse CD16/32 antibody (clone 2.4G2). The cells were then stained with Viability Dye (Thermo Fisher, 65-0865-18), anti-CD45 and Lin-cocktail antibodies including anti-CD3, TCRαβ, TCRγδ, NKp46, CD19, CD11b, CD11c, Ly-6C/G (Gr-1) and Ter119. Viable CD45low/+LinlowYFP− and YFP+ cells were purified at 2 to 1 ratio for adult mice samples. Neonatal sample cells were purified as CD45low/+LinlowCD8−YFP− and YFP+ cells at 2 to 1 ratio. Purified ells were loaded on the Chromium Single Cell Controller (10X Genomics) and libraries were prepped using the Chromium Single Cell 3′ Reagents Kits (v2 chemistry; 10X Genomics), according to the manufacturer's protocol.
GEO Accession
GSM3484669