GSM3996841: ZT23_WT2; Mus musculus; RNA-Seq
Cells were sorted to complete RPMI medium with a purity always greater than 95%. RNA extraction was then performed using the RNeasy Micro Kit (Qiagen) following the manufacturer instructions. Samples were processed following the Smart-Seq 2 guidelines. Briefly, polyA mRNA was initially purified using oligo-dT magnetic beads and then converted to cDNA. Library preparation was achieved using the Nextera XT DNA sample preparation kit (illumina). Libraries were sequenced on a Illumina HiSeq4000 (paired-end, 100bp). Mice were sacrificed with CO2. Small intestines were harvested and thoroughly rinsed with cold PBS1X and had the Peyer patches removed. Intestines were then cut in 1cm pieces, and shaken for 30min in PBS containing 2% FBS, 1% HEPES and 5mM EDTA to remove intraepithelial and epithelial cells. Intestines were then digested with collagenase D (0.5mg/mL; Roche) and DNase I (20U/mL; Roche) in complete RPMI for 30 min at 37 °C, under gentle agitation. Sequentially cells were passed through a 100μM cell strainer and purified by centrifugation 30min at 2400rpm in 40/80 Percoll (GE Healthcare) gradient. Erythrocytes from small intestine were lysed using RBC lysis buffer (eBioscience). Cells were stained and ILC3 were sorted as CD45.2+ Lin- Thy1.2hi KLRG1- cells Libraries were prepared according to Smart-Seq 2 guidelines, using the Nextera XT DNA sample preparation kit (illumina).