GSM5646578: WT-RING1B; Mus musculus; ChIP-Seq
Chromatin preparation was performed as previously described. First, 2×107 cells were crosslinked with 1% formaldehyde for 10 min at room temperature (RT) and then quenched with 0.125 M glycine for another 5 min. Then cells were washed with ice cold PBS and lysed in SDS Buffer (100 mM NaCl, 50 mM Tris-Cl pH8.1, 5 mM EDTA pH 8.0, 0.5% SDS, 1×Protease inhibitor cocktail (PIC)). After spinning, Nuceli were resuspended in appropriate volume of ice-cold IP Buffer (100 mM NaCl, 50 mM Tris-Cl pH8.1, 5 mM EDTA pH 8.0, 0.3% SDS, 1.0% Triton X-100) for sonication using a BioRuptor sonicator (Diagenode). After centrifugation at 16,000 ×g for 20 min at 4°C, the fragmented chromatin was divided into different aliquots that were incubated overnight at 4°C with primary antibodies (RING1B CST 5694s, SUZ12 CST 3737S, RYBP MILLIPORE AB3637). Next, 30 μl protein G magnetic beads were incubated with the reaction for another 3 hrs at 4°C. Then beads were washed 3 times with high salt buffer (1% Triton X-100, 0.1% SDS, 500 mM NaCl, 2 mM EDTA, pH 8.0, 20 mM Tris-HCl, pH 8.0). After reversal of the crosslinking, ChIP DNA was purified for qPCR analysis or library preparation.WT or Asxl2KO cells were washed twice in wash buffer (20 mM HEPES pH 7.5; 150 mM NaCl; 0.5 mM Spermidine; 1×Protease inhibitor cocktail). Meanwhile Concanavalin A beads (Bangs Laboratories, BP531) were activated by washing twice in binding buffer (20 mM HEPES pH 7.5, 10 mM KCl, 1mM MnCl2, 1mM CaCl2), followed by mixing with the cells at RT for 15min. The supernatant was then removed and beads were resuspended in 100 μL antibody buffer (20 mM HEPES pH 7.5; 150 mM NaCl; 0.5 mM Spermidine; 1×PIC; 0.05% Digitonin 2 mM EDTA and 30% BSA). Then 1 μg H2AK119ub1 antibody was added and incubated overnight at 4 ℃. On the next morning, the supernant containing primary antibody was removed and the beads were incubated in 100 μL of Dig-Wash buffer (20 mM HEPES pH 7.5,150 mM NaCl, 0.5 mM Spermidine, 0.0125% Digitonin, 1×PIC) containing the secondary antibody at RT for 1 hr. Then the pellets were washed in 800 μl Dig-wash Buffer for 3 times. Meanwhile 1:200 dilution of pA-Tn5 adapter complex (~0.04 μM) was prepared in Dig-300 Buffer (0.05% Digitonin, 20mM HEPES, pH 7.5, 300mM NaCl, 0.5mM Spermidine,1×PIC). Then the beads were incubated with pA-Tn5 at RT for another 1 hr. After 3 times washing by Dig-300 Buffer, the pellets were resuspended in 50 μL Tagmentation buffer (10mM MgCl2 in Dig-300 Buffer) and incubated at 37 °C for 1 hr. Then Stop buffer (2.25 μL of 0.5 M EDTA, 2.75 μL of 10% SDS and 0.5 μL of 20 mg/mL Proteinase K) was added and incubated at 50 °C for 1 hr. To extract DNA from the reaction, 300 μl hydroxybenzene-chloroform-Isoamyl alcohol were added, thoroughly mixed and centrifuged for 5 min at 16,000 g. The aqueous phase was collected and mixed with 300 μl chloroform. After centrifuging for another 5 min at 16000 g, aqueous phase was then mixed with 750 μl ethanol overnight at 4℃. The samples were centrifuged for 15 min at 16000 g at 4℃. After being washed twice in 1 mL 80% ethanol, the DNA was resuspended in 22 μL TE buffer and used for library amplification (Vazyme). Libraries for both ChIP and Input samples were prepared using TD503 Kit (Vazyme) according to the manufacturer's instruction. The successfully prepared libraries were sequenced as 150 bp paired-end reads on Illumina NovaSeq 6000 platform.