GSM9137092: Uninfected, Treg, cLP; Mus musculus; RNA-Seq
The colon was harvested, cut longitudinally and washed with Hanks' Balanced Salt Solution (HBSS, Gibco) for removing luminal content. Intestinal tissues were incubated twice with HBSS/HEPES supplemented with 5 mM DTT, 5mM EDTA and 5% v/v bovine calf serum (BCS) at 37°C for 15 min, and washed with HBSS/HEPES supplemented with 2% v/v BCS at 37°C for 5 min. Then, the colonic tissues were incubated in RPMI containing 0.167mg/mL liberase TL (Roche), 0.25mg/mL DNase I (Sigma), and 5% BCS on a shaker at 37°C for 30 min. Finally, they were dissociated with gentle MACS dissociator (Mylteni). Immune cells were enriched using a 40- 70% PERCOLL gradient (GE healthcare) and resuspended in FACS buffer (PBS 1X, 2mM EDTA and 2% v/v BCS. Single cell suspensions of the colonic lamina propria collected from 3 mice (age and gender matched littermates) were pooled together per each condition and then sorted on a BD FACSAria cell sorter. Manufacturer's instructions were followed without any significant modifications to capture the cells, generate NGS libraries and perform sequencing. Briefly, cells were captured on a 10x Chromium single cell instrument (10x Genomics) and subjected to single cell barcoded cDNA synthesis using NextGem V3.1 chemistry (10x Genomics). Purified cDNA was amplified and subjected to enzymatic fragmentation, end-repair, A-tailing, adapter ligation and sample indexing to generate sequencing ready libraries. Libraries were assessed by electrophoresis on a BioAnalyzer 2100 instrument (Agilent) and qPCR on a CFX96 (BioRad) instrument prior to sequencing.