GSM5512829: ChIP.07_CTCFWAPL_0h_antiCTCF; Mus musculus; ChIP-Seq
Calibrated ChIP-seq experiments were performed in presence of 10% HEK293T cells as an internal reference using a published protocol (Liu et al., 2021). For chromatin preparation, the mouse embryonic stem cells were mixed with 10% HEK293T cells and cross-linked by a final concentration of 1% formaldehyde for 10 min. The cross-linking reaction was quenched using 2.0 M glycine. The cross-linked cells were then lysed and sonicated to obtain ~300 bp chromatin using Bioruptor Plus sonication device (Diagenode). For ChIP assays, antibodies were first coupled with Protein G beads (ThermoFisher Scientific), and then the sonicated chromatin were incubated overnight at 4oC with the antibody coupled Protein G beads. After over incubation, captured chromatin was washed, eluted and de-crosslinked. The released DNA fragments were purified using MiniElute PCR Purification Kit (Qiagen). The ChIP experiments were performed using the following antibodies: (1) WAPL (16370-1-AP, Proteintech), (2) CTCF (07-729, Merck Millipore), (3) RAD21 (ab154769, Abcam), and (4) H3K4me1 (pAb-037-050, Diagenode). Calibrated ChIP-seq libraries were prepared according to the protocol of KAPA HTP Library Preparation Kit (Roche); 2. RNA-seq libraries were prepared using a TruSeq Stranded RNA LT Kit (Illumina).