title: Illumina HiSeq 2500 paired end sequencing; RNA-seq of mouse small intestinal CCR6+ group3 innate lymphoid cells
design: RNA-seq of mouse small intestinal CCR6+ group3 innate lymphoid cells
library_construction_protocol: ILC3 isolation Isolated small intestine was opened longitudinally and washed with cold HBSS containing 15 mM Hepes, pH 7.2. Tissues were cut in 1-cm pieces and incubated in HBSS buffer containing 10% FBS, 15 mM Hepes, 5 mM EDTA, and 1 mM DTT, pH 7.2, at 37°C two times for 20 min to remove epithelium and intraepithelial lymphocytes. The tissues were digested with 100 U/ml Collagenase VIII (Sigma-Aldrich) in RPMI containing 10% FBS, 15 mM Hepes, 100 U/ml P/S, and 1 mM DTT, pH 7.2, at 37°C in a shaker two times for 1 h. Supernatants were passed through a 70-μm cell strainer and washed in cold HBSS. Pellets were suspended in 90% Percoll, overlaid with 40% Percoll, and centrifuged at 1,800 rpm for 20 min to allow separation of mononuclear cells by density gradient. Interphase was washed and stained with conjugated antibodies.   Cell sorting: ILC3s were sorted as CD45+Lin-NK1.1-CD127+CD117+CCR6+NKp46(GFP)- on a FACS Aria III RNA sequencing: cDNA was prepared using SMARTer Ultra Low RNA kit (Clontech Laboratories) for Illumina Sequencing following the manufacturer's protocol. The Agilent 2100 Bio-analyzer and the High Sensitivity DNA kit were applied to determine the quantity and quality of the cDNA production.
attributes.tag: Experimental Factor: individual
attributes.value: 3