ChIP-seq. ESCs were resuspended in medium, fixated for 10 min at room temperature and lysed as described (Wiehle and Breiling 2016). After nuclei lysis, samples were sonicated 4 × 5 min with 30 s on/off intervals (Bioruptor, Diagenode). SDS content was reduced by dilution as described (Wiehle and Breiling 2016). Chromatin from 6 x 106 cells was immunoprecipitated overnight at 4°C using 5 µg of antibody against CTCF (#61311, Active Motif). Further processing occurred according to instructions of the ChIP-IT High Sensitivity kit (Active Motif). Immunoprecipitated DNA was eluted from Protein A/G agarose beads and purified using the IPure kit (Diagenode). For ChIP-sequencing libraries were generated from 10 ng of DNA using the NEBNext ChIP-Seq Library Prep Master Mix Set (NEB) according to the manufacturer´s instructions. Final libraries were quality controlled and pooled to equimolar amounts and sequenced in 50 bp single-read mode on an Illumina HiSeq 2000 device. Genomic DNA was isolated by lysing freshly prepared cell pellets in pre-lysis buffer (10 mM Tris-HCl (pH 8), 5 mM EDTA, 100 mM NaCl, 1.1% (v/v) SDS, 0.1 mg/ml Proteinase K and 0.04 mg/ml RNAse A) at 37°C overnight. The next day proteins were precipitated by addition of 5 M NaCl and the DNA was isolated by isopropanol precipitation. For transcriptome sequencing total RNA was subjected to DNA digestion using a DNA-Free RNA Kit (Zymo Research). Libraries were prepared from RNA of WT and DKO ESCs using the TruSeq RNA Sample Preparation Kit v2 (Illumina). After quality control libraries were pooled, clustered on the cBot (Illumina) using the TruSeq SR Cluster Kit v3 and sequenced by single-read 50 bp mode on a HiSeq 2000 v3 platform (Illumina) according to Illumina´s instructions. Library preparation for bisulfite sequencing was performed using the TruSeq DNA PCR-Free LT Library Preparation Kit with modifications. Briefly, 2 µg of genomic DNA were fragmented to 150-200 bp using a Covaris ultrasonicator (Covaris, Inc), end-repaired, purified using 1.6x sample purification beads and 3´-adenylated. TruSeq LT adapters were ligated to the DNA fragments followed by bisulfite treatment using the EpiTect Bisulfite Kit (Qiagen) according to instructions of the Illumina WGBS for Methylation Analysis Guide (Part # 15021861 Rev. B). Fragment libraries were enriched with 8 cycles of PCR using KAPA HiFi Uracil+ DNA Polymerase with customized primers (forward: 5'-AATGATACGGCGACCACCGA-3', reverse: 5'-CAAGCAGAAGACGGCATACGAGAT-3') and an annealing temperature of 69 °C according to the settings for paired-end libraries in the technical datasheet (KAPA HiFi HotStart Uracil+ Ready Mix, KR0413 - version 1.12, peqlab). Amplified libraries were purified with 1x Agencourt AMPure XP beads (Beckman Coulter Inc), pooled, clustered on the cBot (Illumina) and sequenced in paired-end mode on a HiSeq2000 platform according to standard Illumina protocols. RNA-seq analysis was performed in Genomatix (Genomatix GmbH). Purified DNA was cloned into Illumina libraries with the NEBNext Ultra library preparation kit (NEB). Illumina NEBNext Ultra ESCs were resuspended in medium, fixated for 10 min at room temperature and lysed as described (Wiehle and Breiling 2016). After nuclei lysis, samples were sonicated 4 × 5 min with 30 s on/off intervals (Bioruptor, Diagenode). SDS content was reduced by dilution as described (Wiehle and Breiling 2016). Chromatin from 6 x 106 cells was immunoprecipitated overnight at 4°C using 5 µg of antibody against CTCF (#61311, Active Motif). Further processing occurred according to instructions of the ChIP-IT High Sensitivity kit (Active Motif). Immunoprecipitated DNA was eluted from Protein A/G agarose beads and purified using the IPure kit (Diagenode).