GSM4089777: HeLa-S3 cells, CTCF_shBHLHE40_HiChIP; Homo sapiens; OTHER
In brief, up to 15 million crosslinked cells were resuspended in 500 μl of ice-cold Hi-C lysis buffer (10 mM Tris-HCl (pH 7.5), 10 mM NaCl, 0.2% NP-40, 1 × Roche protease inhibitors) twice. The nuclei pellet was resuspended in 100 μl of 0.5% SDS and incubated at 62°C for 10 min with no shaking or rotation and then the reaction was quenched with Triton X-100 at 37°C for 15 min. Mbo I restriction enzyme (NEB, R0147) was added to digest the nuclei at 37°C for 2 hr, and heat inactivated at 62°C for 20 min. After filling in the restriction fragment overhangs and marking the DNA ends with biotin, in situ contact was generated with proximity ligation. The nuclei with in situ generated contacts were pelleted at 2,500 × g for 5 min at room temperature and nuclear pellet was resuspended in nuclear lysis buffer (50 mM Tris (pH7.5), 10 mM EDTA, 1% SDS, 1 × Roche protease inhibitor) for sonication and the supernatant was collected by centrifugation at 16,100 × g at 4°C for 15 min. Then the supernatant was transferred to a new tube and diluted with ChIP dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris (pH 7.5), 167 mM NaCl) for ChIP procedures. ChIPed DNA was quantified by Qubit (Thermo Fisher) to estimate the amount of Tn5 (Illumina) needed to generate libraries at the correct size distribution. 150 ng of ChIPed DNA was taken into the BIOTIN capture step and tagmented with Tn5. Tagmented DNA containing beads was PCR amplified and size selected with AMPure XP beads (Beckman). After size selection, libraries were quantified with qPCR against Illumina primers and/or bioanalyzer. Libraries were paired-end sequenced with read lengths of 150.
GEO Accession
GSM4089777