title: GSM3477313: WT_DSGFA_input_DNA; Mus musculus; ChIP-Seq
library_construction_protocol: 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 2 mM DSG (Thermo 20593) for 45 min, and then further cross-linked in 1% formaldehyde for 20 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. To recover the input DNA, the samples were incubated overnight in reverse cross-linking buffer (1% SDS and 0.1 M NaHCO3) at 65 °C. The DNA was purified by QIAquick PCR Purification Kit (QIAGEN) and quantified with Picogreen (Life Technologies). ChIP enrichment was confirmed with qPCR for putative targets prior to library preparation. Rubicon Genomics ThruPLEX DNA-seq Kit (Illumina)
attributes.tag: GEO Accession
attributes.value: GSM3477313