GSM5831927: cChIPseq_GT_wt_CTCF_rep2; Mus musculus; ChIP-Seq
RNA-seq: 5 million mESCs were trypsinized and collected by centrifugation. Resulting cell pellets were washed in 1x PBS and resuspended in 1x DNA/RNA protection reagent (Monarch Total RNA Miniprep Kit, NEB). Subsequently, cells were lysed and total RNA extracted following the mammalian cell protocol including optional on-column DNase I treatment. Calibrated ChIP-seq (cChIP) and ChIPCap-seq: 30 million mESCs and HEK293T cells were collected, washed once in 1x PBS and crosslinked for 7 min in 1% formaldehyde. The crosslinking was quenched by addition of 125 mM glycine and incubated on ice. The crosslinked cells were pelleted by centrifugation for 5 min at 1200g at 4 °C. Nuclei were prepared by washes with NP-Rinse buffer 1 (10 mM Tris pH 8.0, 10 mM EDTA pH 8.0, 0.5 mM EGTA, 0.25% Triton X-100) followed by NP-Rinse buffer 2 (10 mM Tris pH 8.0, 1 mM EDTA, 0.5 mM EGTA, 200 mM NaCl). Afterwards, the nuclei were washed twice with shearing buffer (1 mM EDTA pH 8.0, 10 mM Tris-HCl pH 8.0, 0.1% SDS) and subsequently resuspended in 900 µL shearing buffer with added 1× protease inhibitors complete mini (Roche). Chromatin was sheared by sonication in 15 ml Bioruptor tubes (Diagenode, C01020031) with 437.5 mg sonication beads (Diagenode, C03070001) for 6 cycles (1 min on/1 min off) on a Bioruptor Pico sonicator (Diagenode). For each ChIP reaction 4 % HEK293T-derived human spike-in lysate was combined with mESC lysate and incubated in 1x IP buffer (50 mM HEPES/KOH pH 7.5, 300 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% DOC, 0.1% SDS), with following appropriate antibodies at 4 °C o/n a rotating wheel: H3K27me3 (Diagenode, C15410195), RING1B (Cell Signaling, D22F2), PDS5a (Millipore Sigma #SAB2101764), SUZ12 (Cell Signaling D39F6), H2AK119ub (Cell Signaling D27C4), PCGF1 (Abcam ab202395), RAD21 (Abcam ab992), CTCF (Millipore 070729). Antibody-bound chromatin was captured using Dynabeads protein G beads (Thermofisher #10004D) for 4 hours at 4°C. ChIP washes were performed as described previously (Farcas et al., 2012). ChIPs were washed 5x with 1x IP buffer ( 50 mM HEPES/KOH pH 7.5, 300 mM NaCl, I mM EDTA, 1% Triton-X100, 0.1% DOC, 0.1% SDS), or 1.5x IP buffer for H3K27me3 and H2AK119ub, followed by 3x washes with DOC buffer (10 mM Tris pH 8, 0.25 mM LiCl, 1 mM EDTA, 0.5% NP40, 0.5% DOC) and 1x with TE/50 mM NaCl. ChIP DNA was eluted 2x in elution buffer (1% SDS, 0.1 M NaHCO3 ) at 65°C for 20 min, RNase A treated for 30 min at 37 °C, Proteinase K treated for 3 hrs at 55 °C and crosslinks were reversed o/n at 65 °C. The following day, ChIP samples and corresponding inputs were puriﬁed by PCI extraction and DNA precipitation. In Situ Hi-C: Hi-C was performed as previously described (Rao et al., 2014) with modifications described in (Boyle et al., 2020) as following: 5 million mESCs were crosslinked in 1% formaldehyde for 10 mins before the reaction was quenched by adding 0.2 M final glycine. Cells were permeabilized in lysis buffer (0.2% IGEPAL, 10 mM Tris-HCl pH 8.0, 10 mM NaCl, 1x Halt Protease inhibitor cocktail) and nuclei isolated in NEBuffer 3 supplemented by 0.3% SDS at 62 °C for 10 min. SDS was quenched with 1% Triton X100 at 37°C for 60 min, the nuclei pelleted and resuspended in 250 µl of 1x DpnII buffer with 600 U DpnII (NEB). Following o/n digestion at 37°C, 200 U were added for 2 hrs. DpnII was inactivated for 20 min at 65°C before the DNA ends were filled-in and biotinmarked using Klenow, d(C/G/T)TPs and biotin-14-dATP for 90 min at 37°C. Proximity ligation was performed using T4 DNA ligase (NEB) for 4 hrs at room temperature. Subsequently, nuclei were spun down, resuspended in 200 µl mQ water and digested with proteinase K for 30 min at 55°C in presence of 1% SDS. For crosslink reversal 1.85 M final NaCl was added and samples incubated at 65°C o/n. The next day, sample DNA was ethanol precipitated and sheared in 500 µL sonication buffer (50 mM Tris pH 8.0, 0.1% SDS, 10 mM EDTA) on a Bioruptor Pico sonicator (Diagenode). DNA was then concentrated on Amicon ultra 0.5 30K ﬁlter units (Millipore), biotin-pulldown performed using MyOne Streptavidin T1 beads (Life technologies, 65602) and used for NGS library preparation. RNA-seq: For RNA-seq library preparation, 1 µg of total RNA per sample was enriched for poly-A using the NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB, E7490) and final RNA-seq libraries generated using the NEBNext Ultra II Directional RNA Library Prep kit (NEB, E7760 and NEBNext Multiplex Oligos (NEB, E7335/E7500). cChIP-seq and ChIPCap-seq library preparation: Libraries were prepared using the NEXTflex ChIP-Seq kit (Bio Scientific) following the “No size-selection cleanup” protocol. Libraries were purified using Agencourt AMPure XP (Beckman Coulter) and amplified using the KAPA Real-Time Library Amplification Kit (KAPABiosystems) following the manufacturer's instructions. ChIPCap-seq libraries were prepared identically to ChIP-seq libraries. After PCR amplification the libraries were enriched for loci of interest using the MYbaits kit DNA capture target enrichment system (Arbor Biosciences) according to manufacturer's manual. 120 nucleotides long MYbaits sequence capture probes (Arbor Biosciences) were custom designed against 25 mm9 genomic loci (Table S3). Library quality control including determination of average size and concentration was performed prior to sequencing by commercial Next Generation Sequencing providers. NGS libaries were eventually sequenced as 150 bp paired-end reads on the Illumina HiSeq platform. In situ HiC: DNA ends were repaired, biotin removed from unligated ends and NEXTFLEX DNA barcoded adapters (Perkin Elmer) were ligated. Desired PCR cycle numbers were determined in test endpoint PCRs using Q5 DNA polymerase (NEB M0491L). Final HiC libraries were generated from 4-6 individual PCR reactions, which were pooled and subjected to cleanup and size-selection using AMPure beads (Beckman Coulter A63882). Samples were ﬁrst test sequenced to check library quality before selected Hi-C ibraries were sequenced at greater depth (Table S2). NGS libaries were eventually sequenced as 50 bp single-end reads or 150 bp paired-end reads on Illumina platforms