Illumina HiSeq 4000 sequencing; H3K4me3 occupancy after Rnf20 and Rnf40 knockout in intestinal epithelial cells isolated from mice
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.
Experimental Factor: immunoprecipitate
H3K4me3
Experimental Factor: genotype
Rnf20 knockout