GSM5018593: U2OS cells - LowMYC_rep1_4C; Homo sapiens; OTHER
40 million cells were cross-linked in PBS supplemented with 1% formaldehyde at room temperature for 10 minutes, followed by quenching with 0.25M glycine for 5 minutes. Cells were washed thrice with PBS supplemented with 1mM phenylmethylsulfonyl fluoride (Sigma Aldrich). Nuclei were isolated by lysing cells in 4C Lysis Buffer (10mM Tris-HCl pH 8.0, 10mM NaCl, 5mM EDTA, 0.5% Igepal CA-630) at 4℃ for 10 minutes, followed by homogenization using a dounce homogenizer (Wheaton) (100 strokes using Pestle B). Nuclei were permeabilized at 37℃ with the addition of 0.3% SDS for 1 hour, followed by 2% Triton-X 100 for 1 hour. Primary enzyme digestion with HindIII-HF (NEB) was done at 37℃ for 18h, before proximity ligation in dilute conditions with T4 DNA Ligase (Thermo Scientific) at 16℃ overnight. Crosslinking was reversed with the addition of 55µg/ml Proteinase K (Ambion) at 65℃ for 4 hours and 37℃ overnight, and treated with RNAse A at 37℃ for 1 hour. DNA was purified using phenol/chloroform extraction followed by ethanol precipitation to yield the 3C library. Secondary enzyme digestion with DpnII (NEB) was done on the 3C library at 37℃ overnight, followed by proximity ligation and de-crosslinking as described above. 4C libraries were generated for each viewpoint through nested inverse-PCR using Phusion DNA Polymerase (Thermo Scientific). DNA fragments between 200 and 1000bp were isolated from the 4C libraries by gel excision after running the 4C libraries on a 4-20% gradient TBE gel (Thermo Scientific). The gel slices were shredded and macerated in Tris EDTA buffer at 37℃ overnight and DNA was precipitated using ethanol. The 4C libraries were multiplexed and single-end 1 x 150bp sequencing was performed on the Illumina MiSeq.