GSM8564202: Liver Invariant Natural Killer T cells (iNKT), Diethylnitrosamine (DEN), 38-42 weeks, Hepatocellular carcinoma 3; Mus musculus; RNA-Seq
To isolate hepatic lymphocytes, livers were placed in a 100 μm filter and homogenized with FACS buffer (HBSS, 0.25% BSA, 25 mg/ml DNase I, 0.1% NaN3). Samples were subsequently centrifuged at 1500 rpm for 5 min at 4oC, and pellets were resuspended in 8 ml of Percoll mixture (for 10 ml Percoll mixtures: 0.4 ml 10X PBS, 4 ml Percoll, and 5.6 ml 1X PBS). Samples were centrifuged at 2000 rpm for 20 min at 20oC without braking. Red blood cells were lysed with H2O, and lymphocytes were resuspended in 1 ml FACS Buffer after centrifugation at 1500 rpm for 5 min at 4oC. Lymphocytes were incubated with PBS57-loaded CD1d tetramers  and anti-TCRb for 30 min on ice and in the dark  in the presence of anti-mouse CD16/32 Fc block and propidium iodide. iNKT cells were sorted as CD1dLoaded PBS57 tetramer+TCRb+ cells in a FACSAria III. RNA was extracted using RNAeasy micro kit. QuantSeq-LEXOGEN (3' mRNA-seq Library Prep Kit for Ion Torrent)