GSM3509945: HKC day0 CTCF ChIP-Seq; Homo sapiens; ChIP-Seq
Sample information curated by ChIP-Atlas
TFs and others
Cell type Class
Attributes by original data submitter
CTCF antibody (Millipore #07-729)
Sequenced DNA Library
Chromatin was incubated overnight at 4 °C in 1× incubation buffer (0.15% SDS, 1% Triton X-100, 150 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, and 20 mM HEPES) supplemented with protease inhibitors and 0.1% BSA. A 50:50 mix of Protein A and G Dynabeads (Invitrogen) were added. Antibodies against H3K4me3 (Diagenode, C15410003, 1 μg), H3K27me3 (Diagenode, C15410069, 1.5 μg) and RUNX1 (ab23980, abcam, 4 μg) were used in each ChIP assay. The beads were washed twice with wash buffer 1 (0.1% SDS, 0.1% sodium deoxycholate, 1% Triton X-100, 150 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, and 20 mM HEPES), once with wash buffer 2 (wash buffer 1 with 500 mM NaCl), once with wash buffer 3 (250 mM LiCl, 0.5% sodium deoxycholate, 0.5% NP-50, 1 mM EDTA, 0.5 mM EGTA, and 20 mM HEPES), and twice with wash buffer 4 (1 mM EDTA, 0.5 mM EGTA, and 20 mM HEPES). After the washing steps, beads were rotated for 20 min at room temperature in elution buffer (1% SDS and 0.1 M NaHCO3). The supernatant was de-cross-linked with 200 mM NaCl and 100 μg/mL proteinase K for 4 h at 65 °C. De-cross-linked DNA was purified with MinElute PCR Purification columns (Qiagen). DNA amounts were determined with Qubit fluorometric quantification (ThermoFisher Scientific). The pulled down DNA fragments were proceeded on with library construction using KAPA Hyper Prep Kit (Kapa Biosystems #KK8504) according to the standard protocol. The prepared libraries were then sequenced using the NextSeq 500 (Illumina) according to standard Illumina protocols.