GSM4476747: H3K4me2_MNaseChIP_Villus; Mus musculus; MNase-Seq
Freshly harvested jejunum was flushed with cold PBS, opened longitudinally, cut into 1 cm pieces, and then rotated in 3 mM EDTA in PBS for 5 mins, 10 mins and 40 mins at 4 °C (refresh EDTA/PBS every time). The tissue was then violently shaken to release the epithelium from underlying muscular tissue. The intestine pieces were discard and the supernatant was collected as whole epithelium fraction. 70-μm cell strainer was used to separate the villi and crypt fractions. Villi were collected from the top of a 70-μm cell strainer, while crypts passed through. Cells were pelleted by centrifugation at 170g at 4 °C, and followed by cold PBS wash. For Micrococcal nuclease (MNase) ChIP-seq, mouse villi and crypts were isolated as describe above, and resuspended in digestion buffer (50 mM Tris-HCl pH7.6, 1 mM CaCl2, 0.2% Triton X-100, 5 mM Na butyrate, and protease inhibitor cocktails) and treated with MNase for ChIP-seq, with histone H3K27Ac (Abcam ab4729) or H3K4Me2 (Millipore 07-030) antibodies. NEB Next kit