GSM7566833: ZNF143KO_H3K4me3,ChIP; Homo sapiens; ChIP-Seq
After washed twice by PBS, ~10-20 million cells digested by trypsin and resuspended by 10mL medium. Formaldehyde (Thermo 28908) was added to a final concentration of 1% to cross-link protein and DNA. After incubation at room temperature for 10 minutes, the glycine solution reached a final concentration of 125mM and incubated at room temperature for 5 minutes to quench the cross-linking reaction. Cells were centrifuged at 2,500g for 10 minutes at 4°C to precipitate cells. The cell pellet was washed by ice-cold PBS and quickly frozen by liquid nitrogen. The cell pellet was stored at -80°C until the next step. Cell pellet was resuspended by 1mL ice-cold ChIP buffer 1 (10 mM Tris-HCl pH 7.5, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 150 mM NaCl, 1x protease inhibitors), and lysed twice by ChIP buffer 1 at 4°C for 10 minutes with slow rotation. After centrifuged at 2,500g for 5 minutes at 4°C, the pellet was resuspended by 0.7mL ChIP buffer 1 and incubated at ice for 10 minutes. The cell suspension was sonicated to fragment DNA using Bioruptor Plus Sonicator by high intensity (30 seconds on, 30 seconds rest, 30 cycles), and the DNA size should be 100-10000 bp. After spinning at 14,000g for 10 minutes at 4°C, the supernatant was transferred to a new tube with 50 μL agarose protein A beads (16-157 Millipore) were added. After incubation at 4°C with slow rotation for 1 hour, the supernatant was transferred into a new tube and the primary antibody was added to incubate it at 4°C with slow rotation overnight. 50 μL agarose protein A beads were added and incubated at 4°C with slow rotation for 3 hours. The protein-DNA complexes were sequenced and washed by ChIP buffer 1, ChIP buffer 2 (10 mM Tris-HCl pH 7.5, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 400 mM NaCl), ChIP buffer 3 (10 mM Tris-HCl pH 7.5, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate) and ChIP buffer 4 (50 mM HEPES pH 7.5, 1 mM EDTA, 1% NP-40, 0.7% sodium deoxycholate, 500 mM LiCl). DNA-protein complexes were eluted using 100 μL elution buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA, 1% SDS) by incubation at 65°C for 30 minutes. The above step was repeated again and a total of 200 μL solution of DNA-protein complexes was collected. The DNA-protein complexes solution was mixed with 200 μL TE buffer, and incubated at 65°C overnight to be reversely cross-linked. The DNA-protein complexes were digested by 2 μL RNase A at 37°C for 2 hours and 8 μL proteinase K at 55°C for 2 hours. The DNA was purified using 400μl of phenol-chloroform. The DNA was precipitated by adding 40 μL 3M NaAc pH 5.2, 1.5 μL glycogen (Thermo R0561), and 1mL absolute ethanol, and frozen at -80°C for 30 minutes. After centrifuged at 17,000g for 30 minutes at 4°C, the DNA pellet was washed by 75% ethanol, air-dried, and then solved by 20 μL nuclease-free water. DNA concentration was measured by PicoGreen reagents. 10 ng DNA was used for the library constructed with NEBNext®Ultra™ II DNA Library Prep Kit for Illumina. Library was sequenced on the Illumina HiSeq platform.