GSM4249087: CTCF ChIP-seq SiHa R3; Homo sapiens; ChIP-Seq
Sample information curated by ChIP-Atlas
TFs and others
Cell type Class
Carcinoma Squamous Cell
Attributes by original data submitter
Sequenced DNA Library
CTCF ChIP-seq: We prepared 10 µl of both protein A and protein G beads through three washes of 5mg/mL Dulbecco's phosphate-buffered saline (dPBS) + bovine serum albumin (BSA). We added 10 µl of CTCF antibody (Cell Signalling Technology, Danvers, Massachusetts, Cat No. 2899, Lot 002) to the beads in 300 µl dPBS + BSA and left it to bind for > 6 hours of rotation at 4°C. After incubation, we washed the beads three more times with dPBS + BSA and resuspended in 100 µl of modified Radioimmunoprecipitation assay buffer (RIPA) (10 mmol/l Tris-HCl, pH 8.0; 1 mmol/l EDTA; 140 mmol/l NaCl; 1% Triton X-100; 0.1% SDS; 0.1% sodium deoxycholate) + protease inhibitor (PI). We collected 1 million cells by trypsinization and then fixed for 10 min at room temperature in 300 µl of dPBS + 1% formaldehyde. We added 15 µl of 2.5M glycine after fixing then washed the cells once in PBS + PI before resuspending them in 300 µl of modified RIPA + PI. We sonicated the samples for 32 cycles of 30 sec at full intensity using a Bioruptor Pico (Diagenode, Seraing, Belgium) and pelleted cell debris by spinning at 21130 xG for 15 min. We set aside 15 µl of the supernatant as an input control, and diluted the remaining supernatant with 1700 µl of modified RIPA + PI and 100 µl of washed beads. We incubated the samples at 4°C overnight with rotation. We washed the beads with the following cold buffers in order: modified RIPA, modified RIPA + 500 µmol l−1 NaCl, LiCl buffer (10 mmol l−1 Tris-HCl, pH 8.0; 1 mM EDTA; 250 mmol l−1 LiCl; 0.5% NP-40; 0.5% sodium deoxycholate), and finally twice with TE buffer (pH 8). We resuspended the samples and inputs in 100 µl of de-crosslinking buffer (1% SDS, 0.1000 mol l−1 NaHCO3) and incubated at 65°C for 6 h. We cleaned the samples and inputs using the Monarch PCR & DNA clean-up kit (New England BioLabs, Ipswich, Massachusetts), prepared libraries using the ThruPLEX DNA-seq Kit (Rubicon Genomics, Ann Arbor, Michigan), and size selected to 240 bp–360 bp using a PippinHT 2% Agarose Casette (Sage Science, Beverly, Massachusetts). We sequenced the samples to ∼25 million single-end 50 bp reads each at the Princess Margaret Genomics Core, Toronto, Ontario.