title: GSM7124298: Hudep2_rep1_S2; Homo sapiens; OTHER
library_construction_protocol: 1–2 × 107 cells were fixed for 10 min at room temperature with formaldehyde (Sigma # 252549)  at a final concentration of 2% and quenched with 1 M glycine (Sigma # G7126). After centrifugation, the cell pellet was washed and reconstituted in 1 mL of PBS and permeabilized with 0.005% digitonin (Sigma # D141) for 5–10 min. Permeabilized cells were collected by centrifugation at 500 × g for 5 min and resuspended in low-calcium micrococcal nuclease (MNase) buffer (50 mM Tris-HCl pH 7.5, 1 mM CaCl2). Cells were digested with MNase (NEB # M0247) (7.5 to 50 Kunitz Units) in a reaction volume of 800 µL containing 4 × 106 cells at 37 °C for 1 h on an Eppendorf Thermomixer at 550 rpm. The reaction was quenched by adding ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetra acetic acid (EGTA) (bioWORLD # 405200085) to a final concentration of 5 mM. A 200 µL aliquot was removed as a control to test the digestion efficiency. The reaction mixture was then centrifuged for 5 min at 300 × g, and the digestion buffer was discarded. The cells were resuspended in DNA ligase buffer (Thermo Fisher # EL0013) with 10 mM dNTPs (NEB # N0447L), 5 mM EGTA, 200 U/mL T4 polynucleotide kinase PNK (NEB # M0201L), 100 U/mL DNA polymerase I large (Klenow) fragment (NEB # M0210L), and 300 U/mL T4 DNA ligase (Thermo Fisher # EL0013). The reaction mixture was shaken horizontally on an Eppendorf Thermomixer at 550 rpm (shaking for 15 s then pausing for 30 s) for 2 h at 37 °C then for 8 h at 20 °C. The chromatin was reverse crosslinked with proteinase K at 65 °C (for >4 h), and the DNA was extracted using a Qiagen DNeasy Kit (# 69506). Next, 1–10 μg of DNA from the MNase 3C libraries was sonicated with a Covaris E220 Focused Ultrasonicator (Duty cycle, 10%; intensity, 5; cells per burst, 200; time, 200–300 s). Libraries were prepared using standard reagents from New England BioLabs for Illumina sequencing