Anti rna polymerase

ChIP assays were conducted using the EZ-Magna ChIP™ A/G Chromatin Immunoprecipitation Kit (Millipore). Briefly, chromatin was immunoprecipitated with anti-CARM1 (Abcam) and anti-histone H3R17di-me (Abcam). Anti-RNA polymerase (Millipore) was used as a positive control, and anti-IgG (Millipore) was used as a negative control. ChIP-derived DNA was quantified using real-time PCR with SYBR Green (Applied Biosystems). A total of 4 pairs of primers were designed for the promoter region of each gene in order to detect the enriched genomic DNA fragments. The qPCR values were normalised according to the values of a promoter region of GAPDH. The normalised input signal was defined as 1. The fold enrichment value is indicated as the normalised ChIP signal divided by the normalised input signal.

Hypoxia-treated cells (4 × 10⁶) were cross-linked by 1% formaldehyde treatment. Glycine was used to stop the cross-linking. Chromatin was sonicated and sheared into small fragments following the instructions of the EZ-ChIP™ kit (17-371, Millipore, Billerica, MA). One microgram of anti-RNA polymerase (05-623, Millipore, Billerica, MA), 1 μg of IgG (ab96881, Abcam), or 1 μg of anti-SOX2 antibody (ab79351, Abcam) was added to pull down the target protein. Target protein-bound DNA was purified and subjected to PCR amplification of an ~200 bp fragment of the β-catenin promoter using the following primer sequences: FW: 5′-GCCGAGTGGAAACTTTTGTCG-3′, BW: 5′-GGCAGCGTGTACTTATCCTTCT-3′.