Computational protocol: Super Enhancers and Broad H3K4me3 Domains Form Complex Gene Regulatory Circuits Involving Chromatin Interactions

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Protocol publication

[…] 4C-seq was performed as previously described with some modifications. In brief, 4 × 107 cells were harvested and crosslinked with 1% formaldehyde for 10 min at room temperature with rotation. The crosslinking was quenched by glycine for 5 min at room temperature with rotation. Following SDS and Triton X-100 permeabilization, nuclei were digested with HindIII-HF (NEB) overnight. Following proximity ligation, reverse cross-linking and DNA purification, the circular DNA was digested with DpnII (NEB) 37 °C overnight, and circularized. The 4C-seq library was generated by performing nested inverse PCR using Phusion DNA polymerase (Thermo Scientific) with the primers listed in Table . The cycling condition of the 1st PCR was: 98 g condition of the 1inked with 1% formaldehyde for 10 min at room temperature with rotation. The crosslinking was quenchension at 72 °C for 60 s. 10% of the 1st PCR product was used for the 2nd PCR. The cycling condition for the 2nd PCR was: 98cling condition for the 2 with 1% formaldehyde for 10 min at room temperature with rotation. The crosslinking was quenchension at 72 °C 72 °C for 60 s. The 4C-seq library was purified by 4–20% gradient TBE PAGE gel (ThermoFisher Scientific) and the smear band regions including the expected sizes were excised. The library was recovered by incubating the shattered gel slice with 200 uL TE buffer overnight at 37 °C and the DNA in the supernatant was ethanol precipitated in presence of GlycoBlue (ThermoFisher Scientific). The multiplex 4C-seq library was pooled in equal molar ratio and sequenced on MiSeq (Illumina) with 2X250 bp or 1X150 bp. 500,000–1,000,000 reads were produced for each library. The viewpoint sequence was removed by Tagdust2 and the processed read was mapped by BOWTIE2 and over 80% of each library could be mapped to the hg19 genome. The mapped 4C-seq data was analysed by r3CSeq. […]

Pipeline specifications

Software tools TagDust, Bowtie2, r3Cseq
Application 3C-seq analysis
Organisms Homo sapiens
Diseases Breast Neoplasms, Neoplasms, Leukemia, Myelogenous, Chronic, BCR-ABL Positive