GSM4476744: H3K4me3_HiChIP_Hnf4DKO_Villus_rep2; Mus musculus; ChIP-Seq
Freshly harvested duodenum was flushed with cold PBS, opened longitudinally, cut into 1 cm pieces, and then rotated in 3 mM EDTA in PBS at 4 °C for 5 min, 10 min and 40 min at 4 °C (refresh EDTA/PBS every time). The EDTA incubation time was adjusted to release all the epithelial cells from underlying muscular tissue, so for the WT and mutant tissues, the last incubation step is 40 min and 20 min, respectively. The tissue was then vigorously shaken to release the epithelium, and the supernatant was collected as the whole epithelium fraction; villi were collected from the top of a 70-μm cell strainer while crypts passed through. Cells were pelleted by centrifugation at 170 g at 4 °C and then washed by cold PBS. Duodenal villus and crypt cells were then cross-linked in 1.5% formaldehyde (Sigma F8775) for 10 min at 4°C and then for 50 min at room temperature. Cells were pelleted (300 g, 4 °C for 5 min) and washed with ice-cold PBS twice. Cells were quick frozen in liquid nitrogen and stored at -80°C for later processing for HiChIP. The HiChIP protocol was performed using an antibody to H3K4me3 (Millipore 05-745R, lot 3158071). Phusion High-Fidelity PCR Master Mix (New England Biolabs)