GSM2543153: HeLa WT clone2 ab2; Homo sapiens; ChIP-Seq
Sample information curated by ChIP-Atlas
TFs and others
Cell type Class
Attributes by original data submitter
HeLa cell line, WT, ZBTB48 ChIP
Sequenced DNA Library
Subconfluent HeLa and U2OS cells were washed twice with cold PBS and double cross-linked first with 2 mM DSG (Pierce, predissolved in DMSO) in PBS for 45 min at RT, intermitted by a wash with cold PBS and secondly with 1% formaldehyde in DMEM for 20 min at RT. The formaldehyde was quenched with glycine (0.125 M final concentration) for 5 min at RT, cells were scraped with a plastic scraper and washed once in PBS. For lysis, cells were resuspended in buffer 1 (50 mM Tris-HCl pH 8.0, 250 mM sucrose, 140 mM NaCl, 1 mM EDTA pH 8.0, 10% glycerol, 0.5% IGEPAL-630 (Sigma-Aldrich), 0.25% Triton X-100, 0.25% Tween20) for 15 min, pelleted and subsequently resuspended in buffer 2 (10 mM Tris-HCl pH 8.0, 200 mM NaCl, 1 mM EDTA pH 8.0, 0.5 mM EGTA pH 8.0) for 10 min both at 4 ºC. Sonication was performed in 1.5 ml sonication buffer (1% SDS, 10 mM EDTA pH 8.0, 50 mM Tris-HCl pH 8.0) with a Branson Digital Sonifier S-450D for 7.5 min (30% amplitude, 10 sec on, 20 sec off). Library preparation was performed with the NuGEN Ovation Ultralow Library System V2 1-96. Libraries were prepared with a starting amount of 124-6869pg of ChIP DNA and 10-145ng of input DNA were amplified in 13-18 PCR cycles, depending of the starting material used. Libraries were profiled on a 2100 Bioanalyzer (Agilent) and quantified using the Qubit dsDNA HS Assay Kit on a Qubit 2.0 Fluorometer (Thermo Fisher). Size selection was performed with Lab ChIP XT with XT DNA 700 chips (Perkin Elmer) for the 300-500 bp fragment range according to the manufacturer's instructions. Libraries were pooled in equimolar ratio and sequenced on a HiSeq (Illumina), 50bp SR in high-output mode plus 7 cycles for the index read.