Ctcf antibody

All ChIP-seq experiments were performed using H3K4me3 antibody (Cat # 9751S.
Cell Signaling and Technology, Inc., Danvers, MA, USA), H3K27ac antibody (Cat # 39,133, Active Motif, Carlsbad, CA, USA), CTCF antibody (Cat # 61,311, Active Motif, Carlsbad, CA, USA), H3K27me3 antibody (Cat # 9733, Cell Signaling and Technology, Inc., Danvers, MA, USA), and H3K9me3 antibody (Cat # 13,969, Cell Signaling and Technology, Inc., Danvers, MA, USA) as part of previous studies [31 (link), 44 (link)], and datasets were validated according to ENCODE standards. H3K27ac (ENCSR279KIX) and CTCF (ENCSR460LGH) ChIP-seq data were obtained from ENCODE. H3K4me3, H3K27me3, and H3K9me3 ChIP-seq data were obtained from previous experiments (GSE102616, GSE40050, and GSE118629) (Additional file 1: Table S1). ChIP-seq data were aligned with BWA using hg38, and filtered with Picard (http://broadinstitute.github.io/picard/) and Samtools [49 (link)]. Peaks were then called with MACS2 and were checked for reproducibility with IDR [52 (link)] with FDR at 0.05.

Tissues were extracted from postnatal day 2 (P2) mice and cross-linked in 0.5% formaldehyde in phosphate buffered saline (PBS) and protease inhibitors for 5 min (heart) or 1% formaldehyde for 10 min (brain) and quenched with 0.125 M glycine. Intact nuclei were isolated from tissue digestions and split so that each subsequent reaction had 5 × 10⁶ nuclei. Micrococcal nuclease digestion in 1 mM CaCl₂ was performed [20 U for 1 min] and the reaction was terminated with 2 mM ethylene glycol tetraacetic acid (EGTA), followed immediately by sonication in a 0.1% SDS–PBS solution. Direct ChIP was performed on the soluble nuclear extract at 4°C overnight using either CTCF antibody (Active Motif, Carlsbad, CA, USA) or IgG control antibody (Vector Laboratories, Burlingame, CA, USA). Substrates were pulled down with Invitrogen Protein A Dynabeads. After washing and elution, samples were incubated with Proteinase K overnight at 65°C to reverse cross-links and enriched DNA was purified with phenol–chloroform. Ethanol precipitated DNA was resuspended in 10 mM Tris–HCl solution and analyzed by qPCR using the ActiveMotif qPCR Analysis Kit. Each ChIP assay was performed three times and qPCR results represent the mean ± SEM. ChIP primers are listed in Supplementary Table S1.