Rnap 2 antibody

ChIP was performed as described previously [44 (link)], with the following modifications: chromatin was sheared by using a tip sonicator (Fisher Scientific) for 25 rounds of 20 s pulses with 20 Amplitude and 40 s off. Chromatin was diluted in ChIP dilution buffer (0.01% [w/v] SDS, 1.1% [v/v] Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl pH 8.1, 167 mM NaCl). ChIP was performed overnight at 4 °C using 5 μg RNAP II antibody (8WG16, Millipore), FUS (J2516, Santa Cruz), Ser2 RNAPII (5095, Abcam), with Control IgG (Cell Signaling) and rotated overnight at 4 °C. DNA was isolated after crosslink reversal using a QIAGEN PCR clean up kit prior to qPCR (Table S1).

ChIP was performed using standard protocols (38 (link)) on >1 × 10⁶ cells per immunoprecipitation, with an SMC1A antibody (Bethyl Labs, catalog # A300–055A), a Med1 antibody (Bethyl Labs, catalog # A300–793A), an RNAPII antibody (Millipore, catalog # 05–623) and a phospho-IRF3 antibody (Cell Signaling, catalog # 4947). See Supplementary Table S1 for primer sequences, designed with Primer 3 (39 (link)). Real-time PCR was performed as described above (see 3C), using 500 pg of ChIP DNA per reaction. If the starting sample size was ≥5, and the removal of a single data point reduced the standard error by over 20%, that data point was excluded as an ‘outlier.’ In these instances, no more than one data point was removed.