Anti rnapiis2p

Total cell extracts were obtained from 1×10⁶ cells resuspended in 15μl of RIPA buffer (150mM NaCl, 50mM Tris, pH8, 1% NP40, 0.1% SDS), incubated on ice for 10min, and digested with 1μl of benzonase (Novagen) for 20min at 37°C. Extracts were separated by 10% SDS-PAGE and transferred to nitrocellulose membranes. After 30 min incubation in blocking buffer (TBS, 0.1% Tween 20, 5% powder non-fat milk), membranes were incubated overnight at 4°C with anti-CAL1 (rabbit, 1:1000; from A. Straight), anti-CENP-A (rabbit, 1:1000; Active Motif), anti-FLAG (mouse, 1:1000; Sigma-Aldrich), anti-Dre4 and anti-SSRP1 (rabbit, 1:1000; from S. Hirose), and anti-RNAPIIS2p (mouse, 1:1000; Abcam); anti-CENP-C (guinea pig, 1:3000; (Mellone et al., 2011 (link))); anti-tubulin (mouse, 1:1000; Sigma-Aldrich) or anti-histone H3 (rabbit, 1:5000; Abcam) were used as loading controls.

ChIPs were performed using the MAGnify kit (Life Technologies). 10⁶ cells (~10μg DNA) was used for each IP and chromatin was sheared to fragments 100–300bp long. 1μl of anti-CENP-A (rabbit, Active Motif), anti-SSRP1 (Nakayama et al., 2007 (link)), anti-GFP (Abcam), or anti-RNAPIIS2p (Abcam) were coupled to 10μl beads for 2h and mixed with chromatin overnight at 4°C. Immunoprecipitated DNA was eluted in 50μl of elution buffer, and analyzed by qPCR. Normalization was performed using the formula:
$100\times \text{AE}^(\text{averageCT}\text{INPUT}-\text{averageCT}\text{IP})$ , where AE is the amplification efficiency calculated by the formula
$\text{AE}=10^(-1/\text{slope})$ . The values obtained for induced were normalized by those for uninduced cells to calculate enrichment. For ChIP-seq, DNA from three independent ChIPs were pooled and made into libraries with the TruSeq ChIP kit (Illumina). Samples were ran on a MiSeq using Reagent Kit v3. See supplementary experimental procedures for mapping information.