GSM7847013: HUDEP2_FAM46C_Capture; Homo sapiens; OTHER
10^7 cells were collected, and washed with PBS. We resuspend cells with 9 ml PBS and added 1ml 10% formaldehyde (freshly prepared) for a final concentration of 1%, and incubated 10 min at room temperature on a roller mixer, and added 1.1 ml 2M glycine to quenched the reaction, incubated 10 min at room temperature on a roller mixer and on ice for 5 min. Cells were centrifuged for 10 min, 800g at 4℃, and the cell pellet was resuspended in 500 μl lysis buffer (10 mM Tris-HCl, pH 7.5, 10 mM NaCl, 0.5% NP-40, 1x proteinase inhibitor cocktail) and incubated on ice for 50 min. cells were centrifuged for 10 min, 2500g at 4℃. We then washed nuclear pellet once by 1x NEBuffer, resuspended by 80μl 0.5% SDS, incubated for 10min at 62℃, 900 rpm in thermomixer, and add 4μl proteinase inhibitor，226μl H2O，40μl 10% Triton X-100 to quenched SDS for 15min at 37℃, 900rpm in thermomixer SDS 37℃. For enzyme digestion, we added 40μl 10x NEBuffer, 5μl DpnII (250U) incubating at 37℃, 900 rpm, and added another 5μl DpnII before overnight at 37℃, 900 rpm. After digestion, we deactivated the enzyme at 62℃ for 20 min, cooled the tube and added 96μl 10X ligase buffer，4.8μl BSA（20mg/ml）, 80μl 10% triton x-100，4μl T4 ligase (NEB M202M, 8000 U), 369.6μl H2O, and 5.6 μl proteinase inhibitor, incubated at 16℃, 900rpm for 8h. After ligation, We centrifuged for 10 min at 5000g, and resuspend the pellet with 300 μl proteinase K buffer(50 mM Tris-HCl, pH 7.5, 2.5mM EDTA, 50 mM NaCl, 0.5% SDS) and 3 μl proteinase K(20mg/ml), and incubated at 55℃ overnight. We purified DNA from nuclear via phenol-choloroform extraction and ethanol precipitation, and fragmented DNA to 200 bp via covaries (Duty Factor 10 %, PIP 175 W, 180s, 200 cycles per burst, 4 °C).he DNA library preparation was preformed via KAPA Hyper prep kit (KR0961), and oligonucleotide capture was performed via xGen hybridization capture and wash kit (IDT, 1080584). The DNA library preparation was preformed via KAPA Hyper prep kit (KR0961)