Illumina HiSeq 4000 sequencing; H3K4me3 occupancy after Rnf20 and Rnf40 knockout in intestinal epithelial cells isolated from mice
Upon sacrificing Villin-CreERT2, Rnf20/40wt/wt, Rnf20fl/fl and Rnf40fl/fl mice, colons were isolated and flushed with ice-cold PBS. IECs were isolated from the proximal half of the colon. Colon segments used for IEC isolation were opened longitudinally and ice-cold PBS in a tube by vigorous shaking. Colons were cut into <1 mm² fragments and incubated with collagenase solution (2mg/ml Collagenase type I in washing medium (DMEM/F12 + HEPES supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin)) at 37°C for approximately 30-40 min. To separate crypts from the muscle layer, the suspension was pipetted vigorously up and down using a 1,000 μl pipette. When 50-80% of the crypts have separated from the muscle layer, suspensions were transferred to a tube containing 10 ml washing medium. Upon centrifugation at 250 xg for 5 min, supernatants containing muscle fragments were aspirated and the cell pellets were re-suspended in 10 ml washing medium. The suspensions were divided into different tubes and spun down at 250 xg for 5 min. The supernatant was removed and IEC pellets were snap-frozen in liquid nitrogen.  DNA extraction was performed using Roti(R)-phenol/chloroform/Isoamylalcohol (Roth) followed by precipitation in 100% ethanol and washes with 70% ethanol. Libraries for DNA from ChIP were made using the KAPA Hyper Prep Kit (KAPABiosystems) according to the manufacturer's instructions.