GSM6909631: 4C.NlaIII-DpnII_PAXIP-KO_DMSO_P4HA3_rep1; Homo sapiens; OTHER
Experiments of 4C-seq were performed as previously described (Krijger et al., Methods 170, 17–32 (2020)). Briefly, for each restriction enzyme (RE) combination (RE1 NlaIII, RE2 DpnII; New England Biolabs), replicate and treatment about 10×106 cells were collected, pelleted and cross-linked by 2% methanol-free formaldehyde for 10 minutes. Nuclei were isolated and permeabilized to allow digestion of the chromatin by the primary RE (NlaIII, New England Biolabs). Chromatin fragments were then diluted and ligated before crosslinking reversion. Purified DNA was digested by the secondary RE (DpnII, New England Biolabs) and circularized again by ligation. Re-purified circular fragments were amplified by PCR with View-Point specific primers (see Supplementary Table in corresponding paper) using the Expand Long Template PCR System (Roche). Resulting amplicons were purified with a 0.8x ratio of AMPure XP beads (Beckman Coulter) and amplified using standard indexed Illumina primers as previously described (Krijger et al., Methods 170, 17–32 (2020)) using the Expand Long Template PCR System (Roche). Second-round PCR products were purified with PCR purification columns (Qiagen) and quan-tified by 2100 Bioanalyzer (Agilent, DNA 7500 kit). 4C library was prepared by equimolar mix of the samples. The pooled library was cleaned-up by AMPure XP beads (0.8× ratio) to remove PCR dimers before sequencing through Illumina MiSeq with a 75bp Single-End reads setup.