GSM4816718: cLP_CD4_Maf cKO_1; Mus musculus; RNA-Seq
Colons were first washed for 20min in RPMI medium (GIBCO) with 3% FBS (Sigma-Aldrich), 5mM EDTA (Invitrogen) and 1mM DTT (Sigma) in a shaking incubator at 400rpm at 37℃, followed by three other washes each for 30 seconds by vibrant vortexing in RPMI with 2mM EDTA. The tissue was then cut into little pieces and digested for 30min in RPMI with 100ug/mL Liberase TL (Sigma) and 500ug/mL DNase I (Sigma) Digestion in a shaking incubator at 400rpm at 37℃. Digestion was terminated by addition of ice-cold RPMI with 3% FCS. Cells were washed twice in RPMI with 3% FCS, passed through a 40µm cell strainer and resuspended in ice-cold RPMI with 3% FCS and 1mM EDTA. Live (7-AAD-) TCRβ+CD4+ cells were then sorted by FACS. Cells were processed using Chromium Single Cell 3' Reagent Kits v2 according to manufacturer's protocol (10X Genomics). For each biological replicate, an input of 7,000 single cells was added to an individual channel with a recovery rate of approximately 1,300~2100 cells. The generated scRNA-seq libraries were sequenced using a 75 cycle Nextseq 500 high output V2 kit.
GEO Accession
GSM4816718