GSM3477420: Hnf4gammaKO_Hnf4alphaChIP_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. 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. Standard procedures were used for ChIP on mouse duodenum epithelial cells. Briefly, cells were isolated by 3mM EDTA/PBS and then cross-linked in 1% formaldehyde for 10 min at 4°C and then for 35 or 50 min at room temperature. Cells were washed with ice-cold PBS, resuspended with lysis buffer (1% SDS, 10 mM EDTA pH 8.0, 50 mM Tris pH 8.0, and protease inhibitor cocktails) and further incubated at room temperature for 10 min. Cell lysates were sonicated using a Diagenode Bioruptor to generate 200 bp to 500 bp fragments, as determined by agarose gel electrophoresis. The supernatant of lysates were diluted 8-fold in binding buffer (20 mM Tris pH 8.0, 2 mM EDTA pH 8.0, 150 mM NaCl, 1% Triton X-100, and protease inhibitor cocktails) and incubated with 6 ug of Hnf4alpha (Santa Cruz sc-6556 X, lot #B1015) or Hnf4gamma (Santa Cruz sc-6558 X, lot #F0310) antibody coupled to Dynabeads (Invitrogen) at 4°C overnight. The immunoprecipitates were washed 5 times and rotated at 4°C with RIPA buffer (50 mM HEPES pH 7.6, 1 mM EDTA pH 8.0, 0.7% Na-deoxycholate, 1% NP-40, 0.5M LiCl) and a quick wash with TE buffer (0.1 mM EDTA pH 8.0 and 10 mM Tris pH 8.0). The DNA was recovered by incubating overnight in reverse cross-linking buffer (1% SDS, 0.1 M NaHCO3) at 65 °C. The DNA was purified by QIAquick PCR Purification Kit (QIAGEN) and quantified by using Picogreen (Life Technologies). ChIP enrichment was confirmed with qPCR for putative targets prior to library preparation. Rubicon Genomics ThruPLEX DNA-seq Kit (Illumina)
GEO Accession
GSM3477420