GSM2559436: Villin-Cre+_ infected_18h_Salmonella TCRgd+_2; Mus musculus; RNA-Seq
Intraepithelial lymphocytes were isolated as previously described(Mucida et al., 2007). Briefly, small and large intestines were removed and placed in chilled HBSS media (Gibco) containing 2% FCS. The intestines were carefully cleaned from the mesentery and flushed of fecal content.  Intestines were opened longitudinally and then cut into 1 cm pieces. The intestinal tissue was transferred to a 50 ml Falcon tubes containing 25 ml of cold HBSS complemented with 2% FCS and 5 mM EDTA and shaken (2x) at 230 rpm for 20 min at 37°C. The tissue suspension was passed through a stainless steel sieve into 50-ml conical tubes and the cells were pelleted by centrifugation at 1200 rpm for 10 min at 4°C. The cell pellet was resuspended in complete HBSS, layered over a discontinuous 40/70% Percoll gradient, and centrifuged at 2000 rpm for 30 min. Cells from the 40/70% interface were collected, washed and resuspended in complete RPMI media. These purified cells constituted the intraepithelial lymphocyte (IEL) population.  Cells were sorted using a FACS ARIAII with markers as shown above. Total RNA was extracted using the RNAeasy kit from Qiagen. For total RNA from TCRgd IELs, RNA libraries were prepared using the SMART-Seq™ v4 Ultra™ Low Input RNA kit (ClonTech Labs) . For total RNA from intestinal epithelial cells, RNA libraries were prepared using TruSeq® Stranded mRNA Sample Preparation.  All samples were sequenced using 75 base pair single end reading on a NextSeq 500 instrument (Illumina) at High Output setting.
GEO Accession
GSM2559436