title: ChIP-seq of RNA Polymerase II (Rpb3) from wild-type and Rpb7-QHF cells
design: ChIP-seq of RNA Polymerase II (Rpb3) from wild-type and Rpb7-QHF cells
library_construction_protocol: Overnight cultures of three independent biological replicates of wild-type (WT) and Rpb7-QHF cells were subcultured to an OD600 of 0.2 and grown to an OD600 of ~1. Cells were crosslinked with 1% formaldehyde for 5 min and quenched with 125 mM glycine. After harvesting, cells were flash frozen in liquid N2 and kept at -80°C. Cells were lysed by bead beating in lysis buffer (50 mM HEPES-KOH, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Na-Deoxycholate) and sonicated using a Bioruptor (48 cycles, 10 seconds on, 10 seconds off on a high setting). 10% of cleared lysate was saved as input fraction. Cleared lysate was incubated with anti-Rpb3 (1Y26, abcam) overnight and immunoprecipitated with magnetic protein G Dynabeads for 1 hour. Beads were washed with wash buffer (10 mM Tris-HCl, 250 mM LiCl, 0.5% NP40, 1% Na-Deoxycholate, 1mM EDTA), high salt wash buffer (wash buffer supplemented with 500 mM NaCl) and 1x TE (10 mM Tris-HCl, 1 mM EDTA). Immunoprecipitates were eluted in elution buffer (50 mM Tris-HCl, 10 mM EDTA, 0.5% SDS, 5 mM CaCl2) at 65°C. ChIP eluate and inputs (with 50 μl elution buffer) were incubated with proteinase K at 65°C overnight. DNA was purified using the QIAquick PCR purification kit (Qiagen). Input and ChIP DNA libraries were prepared using the NEBNext Ultra II DNA Library Kit for Illumina. We used the kit parameters optimized for 250 bp inserts. Following library amplification (10 cycles), library concentration and size distribution were assessed using the Agilent 2100 Bioanalyzer instrument (High Sensitivity DNA Assay).