GSM5454445: U937_rep2; Homo sapiens; OTHER
2-5 million U937 cells were cross-linked with 1% v/v formaldehyde for 10 minutes during mixing and reactions were quenched using 0.2M glycine. After washing, cells were lysed for 15 minutes using 300 ul ice-cold 4C lysis buffer (10 mM Tris-HCl pH8.0, 10 mM NaCl, 0.2% Igepal CA630) with 50 μl of protease inhibitors (Sigma, P8340). Pelleted nuclei were washed and resuspended in 50 μl of 0.5% sodium dodecyl sulfate (SDS) and incubated at 62 degrees for 5-10 minutes and reactions were then quenched using 145 μl of water and 25 μl of 10% Triton X-100 (Sigma, 93443). Next, chromatin was digested using DpnII and NlaIII restriction enzymes (New England Biolabs (NEB)) with the following procedure for each enzyme: restriction enzyme was added together with 10X NEB CutSmart and incubated for 2h at 37 degrees with rotation. Reactions were inactivated at 62 degrees, followed by ligation using the ligation master mix (663 μl of water, 120 μl of 10X T4 DNA ligase buffer (NEB), 100 μl of 10% Triton X-100, 12 μl of 10 mg/ml Bovine Serum Albumin (100X BSA), 5 μl of 400 U/ μl T4 DNA Ligase (NEB)) with samples at room temperature for 4h with rotation. Cells were then pelleted at 2,500 g for five minutes, and the supernatant discarded, followed by resuspension in 300 μl buffer (10 mM Tris 0.5 M NaCl, 1% SDS) and addition of 50 μl proteinase K with incubation at 55 degrees for 30 minutes. The temperature was then increased up to 68 degrees overnight. Samples were ethanol precipitated and then purified using 1X Agencourt XP beads (Beckman). To quantify library, quantitative real-time RT-PCR (qPCR) was used to amplify using anchor primers 5' and 3' of the FCGR2A TSS (chr1:161475247; FANTOM5) with Nextera P5 and P7 sequencing adapters (primers: 5':TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGTTACTGTTCTGCTGGCCGGT, 3':GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTCCATTCTGCACCAAACATTCA). The cycle number for the sample to cross threshold in the qPCR was used to determine the number of PCR cycles for library amplification and hence PCR was run for 23 cycles with 100 ng 4C DNA library and NEBNEXT 2X master mix (NEB). Samples were purified using 1X Agencourt XP beads, resuspended in 50 μl Buffer EB, quantified using Qubit (Thermo Fisher Scientific), quality assessed using tape station and finally sequenced using Illumina MiSeq.