Chromatin immunoprecipitation (ChIP) - qPCR and sequencing

Chromatin immunoprecipitation (ChIP) was performed as previously described with some modifications [65]. 100 mg of powdered frozen mouse liver was crosslinked in 1% formaldehyde for 10 min and subsequently quenched with Tris pH 8.0 (250 mM final) for 10 min. The quenched cells were washed twice with ice-cold PBS supplemented with a protease inhibitor cocktail (EDTA-free cOmplete™, Sigma Aldrich), flash frozen in liquid nitrogen, and stored at − 80 °C. Crosslinked cells were thawed on ice and then lysed sequentially on ice and for 10 min at each step in each of the following buffers: LB1 (50 mM HEPES- KOH pH 7.4, 140 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 10% Glycerol, 0.5% NP-40, 0.25% Triton-X-100 and EDTA-free cOmplete™), LB2 (10 mM Tris-HCl pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA and EDTA-free cOmplete™), and SDS shearing lysis buffer (10 mM Tris-HCl pH 8, 1 mM EDTA, 0.15% SDS and EDTA-free cOmplete™). The lysates were sonicated (Bioruptor) at 4 °C to generate DNA fragments of 100–500 bp (3 repetitions of 5 sonication cycles of 30 s on and 30 s off for the CTCF ChIP; 1 repetition of 8 cycles of 30 s on and 30 s off for the H3K9me3 ChIP) and the sonicated lysates were subsequently clarified by centrifugation (15,000 rpm for 15 min at 4 °C). The sonicated lysate was divided into 1.5-mL Eppendorf tubes based on the number of ChIPs performed and topped up to 1 mL with Lysis Buffer 500NaCl (20 mM Tris-HCl, pH 7.5, 500 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1% Triton-X-100, 0.1% Sodium deoxycholate, 0.1% SDS and EDTA-free cOmplete™). This mixture was incubated overnight at 4 °C with beads (Protein A Dynabeads, Invitrogen, for CTCF ChIP; Protein G Dynabeads, Invitrogen, for H3K9me3 ChIP) that had been pre-blocked with 0.5% BSA and mixed with polyclonal CTCF antibody (C15410210–50, Diagenode) or polyclonal H3K9me3 antibody (AB_2532132, Active Motif). To remove non-specifically bound proteins from the CTCF ChIP, beads were washed five times with RIPA Buffer (50 mM HEPES-KOH, pH 7.4, 500 mM LiCl, 1 mM EDTA, 1% NP-40 and 0.7% Sodium deoxycholate) and once with TE Buffer (50 mM Tris-HCl, pH 8.0, 10 mM EDTA) at 4 °C. To remove non-specifically bound proteins from the H3K9me3 ChIP, beads were washed twice with low salt buffer (10 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.15% SDS, 1 mM PMSF) followed by single successive washes with high salt buffer (10 mM Tris-HCl pH 8.0, 500 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.15% SDS, 1 mM PMSF), LiCl buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 0.5 mM EGTA, 250 mM LiCl, 1% NP40, 1% Na- deoxycholate, 1 mM PMSF), and 10 mM Tris pH 8.0 at 4 °C. The DNA-protein complex was eluted from the beads in Elution Buffer (50 mM Tris-HCl, pH 8.0, 10 mM EDTA and 1% SDS) for 20 min at 65 °C, and reverse-crosslinked overnight at 65 °C. The eluted samples were then treated with RNase A (Wako) and Proteinase K (Roche), and purified using a PCR purification kit (NEB).

qPCR was performed with Brilliant II SYBR® Green qPCR Master Mix (Agilent Technologies) in a LightCycler® 480 Instrument II (Roche). The ChIP-qPCR fold enrichments are calculated by 2^ΔCt, where ΔCt is the difference in qPCR Ct value between the tested IAP and a control IAP (either IAP-Dst, IAP-Ell2, or IAP-Asxl3). qPCR primers were designed using Primer-BLAST and are listed in Supplemental Table S3.

ChIP-seq libraries were prepared using KAPA Adapters, KAPA HyperPrep Kit (KAPA Biosystems), and AMPure XP Beads (Beckman Coulter) and quality checked using Qubit, Bioanalyzer, and Tapestation. The libraries were sequenced as 150 bp paired-end reads on the Illumina HiSeq4000. The resulting ChIP-seq data was trimmed by Trim Galore and aligned using bwa 0.7.15. For heatmaps, bamCompare from deeptools 3.3.1 was used to generate CTCF binding scores in 50-bp tiles across the genome, using the combined reads of all eight individuals. Each IAP element is split into five equal-sized tiles. The score is the log2 ratio of ChIP reads to input reads; Integrative Genomics Viewer (IGV) was used to visualise a bedGraph file of the log2 ratios. Using only reads with MAPQ≥10, ChIP peaks and summits were called by MACS2 2.1.0 as described in [80] and were visualized using custom R scripts (see Data Access). Genomic context of CTCF peaks in Additional File 1 was analysed using the ChIPQC package from R/Bioconductor [81]. The MACS2 summits from all 8 CTCF ChIP-seq samples were combined, and the sequence of a 50 bp window centred on each summit (N = 97,746 summits) was used as input to MEME 5.0.4, using the strategy of motif finding from [33]. The FIMO tool from MEME was then used to identify genome-wide locations of the top motif.