Instrument: Illumina HiSeq 1000
Strategy: ChIP-Seq
Source: GENOMIC
Selection: ChIP
Layout: SINGLE
Construction protocol: Promoter Capture Hi-C was carried out with SureSelect target enrichment, using a custom-designed biotinylated RNA bait library for mouse promoters and custom paired-end blockers according to the manufacturer’s instructions (Agilent Technologies) (Schoenfelder et al . 2015 The pluripotent regulatory circuitry connecting promoters to their long-range interacting elements, Genome Res, 25: 582-97) and following the protocol detailed in (Nagano et al. 2015 Comparison of Hi-C results using in-solution versus in-nucleus ligation, Genome Biol, 16: 175). ChIP-seq in EpiSCs was performed according to Schoenfelder et al. 2015, Polycomb repressive complex PRC1 spatially constrains the mouse embryonic stem cell genome, Nat Genet, 47: 1179-86. Cells were fixed in ChIP fixation buffer (1% formaldehyde, 5 μM EGTA, 10 μM EDTA, 1 mM NaCl and 0.5 mM HEPES in PBS) for 10 min at room temperature. Fixation was stopped by the addition of glycine (final concentration of 125 mM). Cells were washed with PBS, buffer A (10 mM HEPES, pH 7.5, 10 mM EDTA, 0.5 mM EGTA and 0.75% Triton X-100) and buffer B (10 mM HEPES, pH 7.5, 200 mM NaCl, 1 mM EDTA and 0.5 mM EGTA). Cells were lysed in lysis buffer (25 mM Tris, pH 7.5, 150 mM NaCl, 5 mM EDTA, 0.1% Triton X-100, 1% SDS, 0.5% deoxycholate and Complete protease inhibitor (Roche)) for 30 min on ice. Sonication was performed using a Biorupter sonicator (Diagenode) to obtain an average DNA fragment size of 300 bp. Promoter Capture Hi-C was carried out with SureSelect target enrichment, using a custom-designed biotinylated RNA bait library for mouse promoters and custom paired-end blockers according to the manufacturer’s instructions (Agilent Technologies) (Schoenfelder et al . 2015 The pluripotent regulatory circuitry connecting promoters to their long-range interacting elements, Genome Res, 25: 582-97) and following the protocol detailed in (Nagano et al. 2015 Comparison of Hi-C results using in-solution versus in-nucleus ligation, Genome Biol, 16: 175). ChIP-seq libraries were prepared using the NEBNext DNA Library Preparation kit, following the manufacturer's instructions. Chromatin was diluted with ChIP dilution buffer (25 mM Tris, pH 7.5, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 0.1% SDS, 0.5% deoxycholate and Complete protease inhibitor). Dynabeads Protein G beads (Life Technologies) were blocked for 1 h at 4 °C with 1 mg/ml BSA and 1 mg/ml yeast tRNA (Life Technologies). For each immunoprecipitation, 150 μg of chromatin and 5 μg of antibody were used. Chromatin was precleared with the blocked beads for 1 h at 4 °C. Chromatin was then incubated with antibody overnight at 4 °C with rotation. Protein-antibody complexes were precipitated by the addition of beads for 2 h. Complexes were washed twice with wash buffer A (50 mM Tris, pH 8.0, 150 mM NaCl, 0.1% SDS, 0.5% deoxycholate, 1% NP-40 and 1 mM EDTA), once with wash buffer B (50 mM Tris, pH 8.0, 500 mM NaCl, 0.1% SDS, 0.5% deoxycholate, 1% NP-40 and 1 mM EDTA), once with wash buffer C (50 mM Tris, pH 8.0, 250 mM lithium chloride, 0.5% deoxycholate, 1% NP-40 and 1 mM EDTA) and once with TE. Samples were treated with RNase A and proteinase K, and cross-links were reversed overnight. DNA was purified using a ChIP DNA clean and concentrator column (Zymo Research). Libraries were prepared using the NEB Next Fast DNA Fragmentation and Library Preparation set for the Ion Torrent kit (E6285S), following the manufacturer's instructions. Briefly, 40 ng of ChIP or input DNA was used for library generation. Libraries were size selected for 250-bp fragments using 2% E-gel Size Select agarose gels (Life Technologies) and were amplified with five PCR cycles. Libraries were sequenced on the Ion Proton Sequencer using Ion PI Chip v2 (Life Technologies). Templates were generated using Ion PI Template OT2 200 kit v3 and Ion PI Sequencing 200 kit v3 or the Ion PI IC 200 kit (Life Technologies).