Anti rna pol 2

Cells were transfected with si-WDR5-2 or control siRNA for 72 h. Chromatin immunoprecipitation was conducted with the EZ-Magna ChIP A/G kit (Millipore) according to the manufacturer's instructions. Briefly, 1 × 10⁶ cells were used for each reaction. Cells were fixed in 1% formaldehyde at room temperature for 10 min, the nucleus was isolated with nuclear lysis buffer (Millipore) supplemented with protease inhibitor cocktail (Millipore). Chromatin DNA was sonicated and sheared to a length between 200 bp and 1000 bp. The sheared chromatin was immunoprecipitated at 4°C overnight with anti-WDR5 (Abcam, ab56919) and anti-H3K4me3 (Abcam, ab8580) antibodies. Normal rabbit IgG was used as a negative control and the anti-RNA pol-II (Millipore) antibody was used as a positive control. The WDR5, H3K4me3 and RNA pol-II protein level in the ChIP assays were detected by Western blotting (Fig S6). The negative control primers were used to detect the DNA fragment out of genes as a distal region control. Primers for ChIP-qPCR are listed in Supplementary Table 3.

The ChIP and Re-ChIP assays were performed as described previously [24 (link)]. Briefly, Cell lysates were prepared, sonicated, and immunoprecipitated with anti-KMT2B (ab104444; abcam), anti-ERα (HC-20X; Santa Cruz Biotechnology, Inc), anti-RNA Pol II (05-623B; Millipore) or anti-H3K4me1 (07–436; Millipore) antibodies. The protein-DNA cross-linking of the immunoprecipitated complexes were reversed and the DNA was extracted for subsequent Real-Time qPCR analysis. The primer sets for the qPCR are listed in S2 Table. For the Re-ChIP experiments, after the initial ChIP with the first antibody, the protein-DNA complexes were eluted by incubation for 30 min at 37°C in 25 μl 10 mM DTT. After centrifugation, the supernatant was diluted 20 times with Re-ChIP buffer and subjected again to the ChIP procedure with the second antibody.

Anti-RelB (CAT# 10544), anti-p52 (CAT# 37359), and rabbit anti-Flag (CAT# H2368) antibodies were purchased from Cell Signaling Technology. Mouse anti-Myc (CAT# 05-724), anti-p24 (CAT# MAB880-A), anti-RNA Pol II (CAT# 05-623B), normal mouse IgG (CAT# 12-371B) and normal rabbit IgG (CAT# 12-370) were from Millipore. Mouse anti-Flag (M2) (CAT# F1804), mouse anti-HA (CAT# H3663), and rabbit anti-EGFP (CAT# G1544) antibodies were obtained from Sigma. Anti-GAPDH (CAT# sc-32233), anti-Tubulin (CAT# sc-32293), rabbit anti-HA (CAT# sc-805), rabbit anti-Myc (CAT# sc-789), mouse anti-EGFP (CAT# sc-9996) and horseradish peroxidase-conjugated secondary antibodies (CAT# sc-2005 and sc-2004) were from Santa Cruz Biotechnology. FITC-conjugated goat anti-mouse IgG (CAT# 115-095-005) and TRITC-conjugated goat anti-rabbit IgG (CAT# 111-025-003) were from Jackson Immuno Research. RNase A (CAT# 20-297) was purchased from Millipore. DAPI (4′,6-diamidino-2-phenylindole) (CAT# D8417) and Phorbol 12-myristate 13-acetate (PMA) (CAT# P8139) were purchased from Sigma.

ChIP analysis was performed using a ChIP Assay Kit (Millipore) following the manufacturer’s instructions. Briefly, 1 × 10⁶ cells were used for each reaction. Cells were fixed in 1% formaldehyde at 37 °C for10 min, and nuclei were isolated with nuclear lysis buffer (Millipore) supplemented with a protease inhibitor cocktail (Millipore). Chromatin DNA was sonicated and sheared to a length between 200 bp and 1000 bp. Sheared chromatin was immunoprecipitated at 4 °C overnight with anti-H3K9ac (9649, Cell Signaling Technology), anti-H3K27ac (ab3594, Abcam), anti-H3K27me3 (9733, Cell Signaling Technology),  or anti-RBBP4 (NBP1-41201, Novus). IgG was used as a negative control and anti-RNA polII (Millipore) served as a positive control antibody. Protein A/G bead-antibody/chromatin complexes were washed with low salt buffer, high salt buffer, LiCl buffer, and then TE buffer to eliminate nonspecific binding. Protein/DNA complexes were reverse cross-linked, and DNA was purified using NucleoSpin^®. Levels of ChIP-purified DNA were determined by qPCR (see Supplementary Table 4 for primer sequences). Relative enrichment of the indicated DNA regions were calculated using the Percent Input Method according to the manufacturer’s instructions and are presented as % input.