GSM2044564: THP1 POLR2A 4C; Homo sapiens; OTHER
40 million 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, R3104L) overnight. Following proximity ligation, reverse cross-linking and DNA purification, the circular DNA was digested with DpnII (NEB, R0543S) 37 ˚C overnight, and circularized. 4C-seq was performed as previously described with some modifications (Splinter et. al. Methods 58 (2012) 221-230). The 4C-seq library was generated by performing nested inverse PCR using Phusion DNA polymerase (Thermo Scientific, F-531) with the primers listed in Table S3. The cycling condition of the 1st PCR was: 98˚C for 30s, 98˚C for 5s, 56˚C for 30s, 72˚C for 45s and repeat Step 2 to 4 for 15 times and the PCR end with a final extension at 72˚C for 60s. 10% of the 1st PCR product was used for 2nd PCR. The cycling condition for 2nd PCR was: 98˚C for 30s, 98˚C for 5s, 60˚C for 30s, 72˚C for 45s and repeat Step 2 to 4 for 14 or 16 times and the PCR end with a final extension at 72˚C for 60s.  The 4C-seq library was purified by 4-20% gradient TBE PAGE gel (ThermoFisher Scientific, EC6225BOX) and the smear band regions including expected size was excised. The library was recovered by incubating the shattered gel slice with 200uL TE buffer overnight at 37˚C and the DNA in supernatant was ethanol precipitated in presence of GlycoBlue (ThermoFisher Scientific AM9516). The multiplex 4C-seq library was pooled in equal molar ratio and sequenced on Mi-seq with 2X250bp or 1X150bp. The multiplex 4C-seq library consisted of 3 cell lines (HL60, THP1, K562) and 2 viewpoints (MYC, POLR2A) with 2 replicates per condition. 500,000-1,000,000 reads were produced for each library.
GEO Accession
GSM2044564