Gtx35035

ChIP assays were performed as described^{26 (link)}. Complexes were immunoprecipitated overnight with 2 μg of antibodies specific for SMYD3 (GTX121945, GeneTex), rabbit IgG (GTX35035, GeneTex), H3 (ab1791, Abcam, Cambridge, MA, USA), H3K4me3 (ab10158, Abcam), and RNA polymerase II CTD repeat YSPTSPS (phosphor Ser5) (ab5131, Abcam). Input samples were processed in parallel. Antibody/protein complexes were collected by 40 μl of protein G-coupled Sepharose beads (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA) and washed as follows: once with Tris/EDTA-150 mM NaCl, twice with Tris/EDTA-500 mM NaCl, and once with PBS. Immune complexes were eluted with 1% SDS and TE buffer. After decrosslinking, DNA was purified using a PCR cleanup kit (Qiagen) and analysed by qRT-PCR. The results were expressed as the percentage of the initial inputs. The primer sets used for the ChIP assay are listed in Supplementary Table S2.

ChIP assays for CTCF, RNAP II, H3K4me3, and H3K9me3 were performed using 3 × 10⁶ B95-8 cells, SNU719 cells, and BART(+/-)·S13 (BART(+)·S13⁺, Mt BART(+)·S13^-, BART(-)·S13⁺, Mt BART(-)·S13^-) HEK293-EBV cells per sample, according to the crosslinking chromatin immunoprecipitation (X-ChIP) protocol provided by Abcam (Cambridge, UK) with a slight modification. Before crosslinking, we induced EBV lytic reactivation by treating these cells with TPA (20 ng/mL) and NaB (3 mM) for 48 h. According to the manufacturer’s protocol, a Bioruptor (BMS, Korea) was used to sonicate the genomic DNA. Sonicated cell lysates were subjected to immunoprecipitation with antibodies against CTCF (07–729, Millipore), RNAPII (ab26721, Abcam), H3K4me3 (07–442, Millipore), H3K9me3 (07–472, Millipore), and normal rabbit IgG (GTX35035, Genetex). The precipitates were incubated with ChIP elution buffer (1% SDS, 100 mM NaHCO₃); next, samples were reverse-crosslinked at 65°C overnight and purified using Promega columns. Purified DNA was analyzed using RT-qPCR. ChIP DNA values were calculated as a proportional expression over isotype-specific input DNA values of each primer set.