NextSeq 500 paired end sequencing; RNA-seq analysis of colonic intestinal epithelial cells (IECs) isolated from naive wild-type C57BL/6 mice, DSS-treated wild-type C57BL/6 mice, Il10-/- C57BL/6 mice and Il10-/- C57BL/6 mice with IEC-specific Rabgef1 deletion
RNA-seq analysis of colonic intestinal epithelial cells (IECs) isolated from naive wild-type C57BL/6 mice, DSS-treated wild-type C57BL/6 mice, Il10-/- C57BL/6 mice and Il10-/- C57BL/6 mice with IEC-specific Rabgef1 deletion
To obtain colonic IECs, entire colons were flushed with PBS to empty the contents, opened longitudinally and rinsed 3 times in PBS containing 2% FBS. Such pieces were shaken during 40 minutes at 4°C in PBS with 2% FBS and 3 mM EDTA. The cell suspension obtained was filtered on a 70 µm cell strainer and stained for FACS. Cell sorting was performed on a FACSARIA III (BD Biosciences, Franklin Lakes, USA). Staining reactions were performed at 4°C after incubation with 2.4G2 Fc receptor blocking antibodies to reduce non-specific binding. FSC-W and FSC-A discrimination was used to exclude doublet cells. Colonic IECs were selected as CD45-EPCAM+ cells. For RNA sequencing, each biological replicate contained IECs isolated and pooled from 2-3 mice. Post-sorting viability and purity were above 95% for each sample. RNA was isolated using RNeasy mini kit (Qiagen, Hilden, Germany). RNA levels were determined using a NanoDrop™ (ThermoFisher Scientific, Waltham, USA). The quality of RNA samples was checked using Bioanalyzer RNA Nano (Agilent RNA 6000 Nano Kit, Agilent Technologies, Santa Clara, USA).  Libraries were prepared using the TruSeq mRNA Stranded preparation kit (Illumina, San Diego, USA). Libraries were quality-checked on the QIAxcel (Qiagen, Hilden, Germany) and quantified using the Kapa kit (Roche, Basel, Switzerland). Libraries were then normalized and pooled.