GSM8795422: PDAC_cell,SMARCA5; Mus musculus; ChIP-Seq
Cells were resuspended in 6mL DMEM and 375ml 16% Formaldehyde (w/v) (Pierce, 28908) was added at room temperature for 8 min with gentle shaking. For inactivation, 319 mL 2.5M Glycine was added. Cells were washed with cold PBS twice and centrifuged at 400g for 5 min 3mL ChIP lysis buffer (50 mMHEPES/KOH pH 7.5, 140 mM NaCl, 0.1% sodium deoxycholate, 1% Triton X-100, 1 mM EDTA, 0.1% SDS, 0.25% sarkosyl and proteinase inhibitor cocktail) was added into the cell precipitate. After sonicated, the supernatant was collected by centrifugation at 13000g for 10min. The supernatant was diluted with dilution buffer (50 mM HEPES/KOH pH 7.5, 140 mM NaCl, 1% Triton X-100, 1 mM EDTA and 13proteinase inhibitor cocktail). DNA was isolated from supernatant by IP with antibodies . IPed DNA was washed with high salt buffer (20 mM Tris pH 7.9, 500 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1% SDS and 13proteinase inhibitor cocktail) 7 times and TE buffer (1M Tris-HCl pH 8.0, 0.5M EDTA) once. After treated with RNase A (Qiagen, 19101) and proteinase K (TIANGEN, RT403), transfer DNA was purified using phenol/chloroform (pH 7.0) and ethanol precipitation. The End-It DNA End-Repair Kit (Lucigen, ER81050) was used to convertpurified DNA to 50-phosphorylated, blunt-end DNA. After treated with Klenow Fragment (3'/50 exo-), barcodes (BIOO Scientific, 514120) were ligated with Quick Ligation kit (NEB, M2200). After size selection, 15 cycles of PCR were used to amplify the library.