H3k9me2 antibody

ChIP assays were performed using Pierce Agarose ChIP Kit (#26156, Thermo) according to the manufacturer’s protocol. In brief, after transfection for 72 h, 1% formaldehyde was added to the medium and porcine GCs were cross-linked for 10 min, and then under 6U of micrococcal nuclease had been added in a 37 °C water bath for 15 min. H3K4me2 antibody (#9725 S), H3K4me3 antibody (#9751 S), H3K9me2 antibody (#4658 S) and H3K9ac antibody (#9649 S) were all obtained from Cell Signaling Technology (USA), and were used to pull down the immunoprecipitated complex. After pull down, the fragments of interest were detected by PCR and the amplified products were analyzed by 3% agarose gel electrophoresis, and the enrichment was normalized to the IgG group. IgG antibody (#2985 S, Cell Signaling Technology) was used as a negative control and unprocessed DNA served as the input control. The primers used for the ChIP assay are listed in Table S5.

CUT&Tag assays were performed as described previously with some modifications (50 (link)). Briefly, 1 × 10⁵ cells were washed twice with wash buffer [20 mM HEPES (pH 7.5), 150 mM NaCl, 0.5 mM spermidine and 1× protease inhibitors] by gentle pipetting. A 10 μl aliquot of concanavalin A-coated magnetic beads (Bangs Laboratories, BP531) was activated and added per sample and incubated at RT for 10 min. The supernatants were removed and bead-bound cells were resuspended in 100 μl of wash buffer containing 0.01% digitonin and 2 mM EDTA. The H3K9me2 antibody (Cell Signaling Technology, 4658) was added and incubated overnight at 4°C on a rotator. The beads were washed and resuspended in 300-wash buffer containing 300 mM NaCl with pG-Tn5 (Vazyme, S602) at RT for 1 h. The beads were washed and tagmentation was performed in 300-wash buffer supplemented with 10 mM MgCl₂ at 37°C for 1 h. To stop the tagmentation reaction, 2.25 μl of 0.5 M EDTA, 2.75 μl of 10% SDS and 5 μl of proteinase K (20 mg/ml) were added and further incubated at 55°C for 2 h. Then the genomic DNAs were extracted by phenol–chloroform and subjected to PCR amplification using NEBNext High-Fidelity 2× PCR Master Mix (NEB, M0541S) for 13 cycles. The libraries were size-selected with 1.2× AMpure XP beads (Beckman Coulter, A63881) and sequenced on a NovaSeq 6000 sequencer.