NextSeq 500 sequencing; RNA-seq of mouse Lgr5 expressing intestinal stem cells (ISCs) and transit amplifying (TA) cells
RNA-seq of mouse Lgr5 expressing intestinal stem cells (ISCs) and transit amplifying (TA) cells
Mouse small intestines were flushed with cold PBS, opened and mucus was removed. Intestine was cut to small fragments and incubated with several changes of 10 mM EDTA in PBS on ice for 2 h. Epithelium was detached by vigorous shaking. To enrich crypts, tissue suspension was filtered through 70 μm nylon mesh. Enriched crypts were washed once with cold PBS and plated into 60% Matrigel (BD Biosciences) with ENR media. 10 μM Y-27632 was added to the media for the first 2 days. To isolate single cells, isolated crypts were dissociated in TrypLE Express (Gibco) with 1000 U/ml of DNaseI (Roche) +32 °C for 90 seconds. Cells were washed and stained with antibodies anti-CD31-PE (Biolegend, Mec13.3), anti-CD45-PE (eBioscience, 30-F11), anti-Ter119-PE (Biolegend, Ter119), anti-EpCAM-APC (eBioscience, G8.8) and anti-CD24-Pacific Blue (Biolegend, M1/69). Finally, cells were resuspended to SMEM media (Sigma) supplemented with 7-AAD (Life) for live cell separation. Cells were sorted by using FACSAria II or FACSAria Fusion (BD Biosciences). Total RNA from sorted cells were isolated by Trizol purification. Samples were first treated with HL-dsDNAse (ArticzymesP/N 80200-050) to remove residual DNA. An Ovation Universal RNA-Seq System kit was used for Illumina library preparations (NuGEN Technologies Inc., CA, USA). Purified total RNA (100 ng) was used and primers for ribosomal removal were designed and used as outlined in the kit manual. Libraries were purified with AMPure XP beads (Beckman Coulter Inc., MA, USA),