title: Illumina HiSeq 2500 sequencing; RNA libraries of sorted CD8+ memory T cells from the spleen and RNA libraries of sorted IELs from the small intestine of C57BL/6J mice
design: RNA libraries of sorted CD8+ memory T cells from the spleen and RNA libraries of sorted IELs from the small intestine of C57BL/6J mice
library_construction_protocol: Intestinal IELs were isolated by longitudinal opening of the small intestine, 0.5 cm fragments were shaken for 30 min at 37 ̊C in PBS supplemented with 10 % FCS, 10 mM EDTA, 20 mM HEPES, 1 mM sodium pyruvate, 10 µg/ml Polymyxin B. IELs were put through a 100 µm strainer and washed. Single-cell suspensions were further purified using 37.5 % isotonic Percoll. Spleen cells were mashed through 70 µm strainer, cells were lysed with red blood cell lysis buffer. CD8+ T cells were purified by AutoMACS (Miltenyi) and sorted with BD Influx (IELs CD8α+, TCRβ-; Memory: CD8α+CD44hi). Viability of the sorted cells was over 95 %, using DAPI staining to exclude dead cells. RNA isolation was preformed using Qiagens RNeasy Mini Kit (#74104) following the manufactures instructions. The RNA Integrity Number (RIN) was determined by running the samples on an Agilent Bioanalyser RNA Pico Chip (#5067-1513). The RNA content was determined on a NanoDrop spectrometer. All samples were used for RNA sequencing based on their RIN. An input of 300 ng RNA per sample was used for the generation of unidirectional RNA libraries using TruSeq RNA Sample Preparation V2 LT from Illumina (#RS930-1021), following the manufactures instructions. During RNA library preparation AMPureXP beads were used, Agencourt AMPureXP Reagent (#A63881) Beckman Coulter. The quality of RNA libraries was assessed on Agilent Bioanalyser High Sensitivity DNA Chips (#5067-4626).