GSM3280822: DSS_19083_05_merged; Mus musculus; RNA-Seq
Mouse intestines were opened longitudinally, washed with PBS and villi were scraped away. The intestinal tissue was then dissected in 5-10 mm long pieces and washed 3 times with PBS. Whole crypts were extracted from the small intestine by incubating the minced tissues with 2 mM EDTA for 30 min. at 4°C. Crypts were then pelleted and passed through a 70 μm strainer. Isolated crypts were first digested to single cells after incubation with TrypLE (Life Technologies) and 2000 U/ml-1 Dnase (Sigma) for 10 min. at 37°C. Dissociated cells were passed through a 40 μm strainer, stained with an APC-conjugated CD24 antibody (clone M1/69 BioLegend), and, to exclude non-epithelial cells, with the following Ab's for 30 min. at 4°C: BV421-conjugated TER119 (Biolegend), CD31 (Biolegend), CD45 (Biolegend). For Paneth cell analysis, an additional staining with a PE-conjugated CD117 antibody (Biolegend) was also performed. Cells were then sorted by FACS (BD FACSAriaIII; BD Bioscience). Single viable epithelial cells were gated by CD24 and side scatter (SSC), and by negative selection against the above BV421-conjugated Ab's and with DAPI to discriminate live from dead cells. Detailed FACS gating strategy for the small intestine was performed as previously described (Roth et al., 2012). RNA was isolated from isolated Paneth cells using TRIzolTM Reagent (ThermoFisher Scientific) according to the manufacturer's instructions. RNA quality and quantity was evaluated on a 2100 Bio-analyzer (Agilent) using the Agilent RNA 6000 Pico Kit (Agilent Technologies). For RNA sequencing analysis, RNA samples were further processed by GATC-Biotech (Konstanz, Germany) according to the INVIEWTM Transcriptome Explore library preparation protocol (Illumina based). RNA libraries were prepared for sequencing using standard Illumina protocols