H2AK119ub1 ChIP-seq of human:LHCN-M2 Cells-SMCHD1KO1
The cells were corsslinked with with 1% formaldehyde for 10 min at room temperature and quenched by 125 mM glycine. The crosslinked cells were then lysed in lysis buffer (50 mM HEPES-KOH, pH 7.9, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP40, 0.25% Triton X-100, supplemented with cOmplete protease inhibitors (Roche)) and incubated on ice for 10 min. The cell pellets were then washed with wash buffer (10 mmol/L Tris-Cl, pH 8.1, 200 mM NaCl, 1 mM EDTA, pH 8.0, 0.5 mmol/L EGTA, pH 8.0, supplemented with cOmplete proteinase inhibitors) and shearing buffer (0.1% SDS, 1 mM EDTA, 10 mM Tris-Cl, pH 8.1, supplemented with cOmplete proteinase inhibitors). Then, the cell pellet were resuspended in shearing buffer and sonicated with Covaris E220 Evo sonicator to shear DNA from 300 to 500 bp size fragments. The resulting lysate was supplied with NaCl and Triton X-100 to reach a final concentration at 150 mM NaCl and 1% Triton X-100, then cleared by centrifugation for 10 min at 20,000g. 25 ug chromatin were then incubated with washed Dynabeads Protein G (Invitrogen, 10004D) and antibody overnight on a rotator at 4C. Then, Beads were collected and washed  with low salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mMHEPES-KOH, pH 7.9, 150 mM NaCl supplemented with cOmplete proteinase inhibitors), high salt buffer (0.1% SDS, 1% Triton X-100, 2 mMEDTA, 20 mM HEPES-KOH, pH7.9, 500 mM NaCl supplemented with cOmplete proteinase inhibitors) and LiCl buffer (100 mM Tris-Cl, pH 7.5, 500 mM LiCl, 1% NP40, 1% sodium deoxycholate) twice and with TE buffer (10 mmol/L Tris-Cl, pH 8.1, 1 mmol/L EDTA) once. ChIP DNA were eluted with Protein K digestion buffer(20mM Hepes pH7.9, 1mM EDTA, 0.5% SDS) and 1ul of Proteinase K (20mg/ml) .Purified DNA was quantified for library preparation with  Qubit sensitivity dsDNA HS Kit. Libraries then were prepared using the TruSeq ChIP Sample Preparation Kit (Illumina, IP-2021012, IP-2021024) according to the manufacturer's instruction. Briefly, 5 ng of ChIP-DNA was used for  input and IP samples. Libraries were amplified by 14 cycles on the thermocycler. Libraries then were quantified and validated using the Agilent High Sensitivity DNA Kit and bioanalyzer. Sequencing was performed with an Illumina HiSeq  with 150-bp paired-end read runs.