title: GSM3593259: HiC_teloHAEC_4h_r2; Homo sapiens; Hi-C
library_construction_protocol: In situ Hi-C libraries were prepared as previously described (Belaghzal, Decker and Gibcus, 2017) with the following specifications and modifications:  Approximately 8x10^6 cells per sample were crosslinked (PFA 1%), pelleted and washed in ice-cold PBS prior to lysis and chromatin digestion with DpnII. Biotin-14-dATP fill-in and blunt end ligation was performed as described. Reverse crosslinking was performed in two subsequent 16 and 2 hours incubations with 500 μg of proteinase K prepared at 10 mg/mL in 5mM Tris-HCl pH 7.5, 50% glycerol, 1mM CaCl2 for each step. DNA purification was performed using 15mL MaXtract High Density tubes (Qiagen) with subsequent biotin removal from un-ligated ends using T4 DNA polymerase. For NGS preparation, between 20 and 40 μg of purified Hi-C DNA was used as starting material for all subsequent steps. Sonication to 200-300 bp fragments was carried in an S2 Focused-ultrasonicator with no alterations to the suggested parameters. 300-500 bp fragments were purified with AMPure XP beads and end-repaired as described. Biotin pulldown was performed with 200 μg of Dynabeads MyOne Streptavidin C1 (Invitrogen) per sample. A-tailing was performed as described. Illumina adapters were ligated to 3' d-ATP overhangs using T4 DNA ligase. Production PCR was carried out with 9 cycles of PCR to obtain sufficient quantity for NGS while limiting PCR duplicates. Primers and primer dimers were removed by purification AMPure XP beads.
attributes.tag: GEO Accession
attributes.value: GSM3593259