GSM6709937
Cells were cross-linked with 1% formaldehyde for 10 min at room temperature. Glycine was added to 75mM quenched formaldehyde, then incubated with RT for 5min and rinsed twice with PBS. Cells were added with 1ml PBS and collected into EP tubes with a scraper. The cells were rotated at 1400g at 4℃ for 5min. The supernatant was discarded and re-suspended with 1mL LB1(50mM HEPES pH= 7.5, 140mM NaCl, 1mM EDTA, 10% glycerol, 0.5%NP-40, 0.25% Triton X-100). The cells were incubated on a rotating wheel at 4°C for 10min. Cells were centrifuged at 1400g for 5min at 4 ° C, then resuspended with 1mL LB2 (10mM Tris-HCl pH=8.0, 200mM NaCl, 1mM EDTA, 0.5mm EGTA) and incubated on RT rotating wheels for 10min. Cells were centrifuged at 1400g for 5min at 4 ° C and resuspended in 1mL buffered 1mL LB3 (10mM Tris-HCl pH=8.0, 100mM NaCl, 0.5mM EDTA, 0,5mM EGTA, 0.1% Na-Deoxycholate, 0.5%N-lauroylsarcosine). Cells were placed on ice for 10min and then transferred to ultrasound tube. Ultrasonic processing was performed using Bioruptor :30 seconds on - 30 seconds off, 10 cycles, a total of 20 cycles at high power, resting on ice for 10min every 10 cycles. The sonicated chromatin was centrifuged at full speed for 10min at 4°C. To check ultrasonic efficiency, 10mL samples were back-cross-linked by the addition of 40mL ChIP elution buffer (10mM Tris pH8, 300mM NaCl, 5mM EDTA, 0.5% SDS). DNA was cultured at 65°C for 12h, and 1mL of RNase A (10mg/mL) was added to the sonicated DNA and incubated at 37°C for 1h. Finally, 5mL proteinase K (20mg/mL) was added and incubated for 2h at 55°C. DNA size distribution was examined on 1.5% agarose gel.  Take 10% supernatant as input, mix proteinG beads (Invitrogen, #1004D) in advance30ul, add 1ml block solution blocking solution (0.5%BSA, IgG-free, Protease-free, ImmumoReasearch #1001-000-062) into EP tube, then centrifuge vortex, place on magnetic magnetic frame, wait for 1min, wash three times. Add 5ug of antibody into beads, rotate at 4℃ for 8h, and resuspended with 50ul blocking solution. Add 50ul antibody /beads mixture into the cleavage product and rotate overnight at 4℃. Then Wash Buffer(50mM Hepes-KOH, pH 7.6, 500mM LiCl, 1mM EDTA, 1%NP-40, 0.7%Na-Deoxycholate) was washed and resuspended with Elution Buffer(50mM Tris-HCl, pH 8.0, 10mM EDTA, 1%SDS ), RNA and protein were digested, DNA was extracted, resuspended with 10ul water, quantitative concentration (≥0.2ng/ul), and 1ng was used on machine. DNA integrity was determined via bioanalyzer (Agilent) using standard  Pico chip. The libraries of samples for unstranded sequencing were constrcuted by TruSeq RNA Library Prep Kit and sequenced on Illumina NextSeq 500.