Name: Pol2_Rsc8-Myc_444_p
Instrument: NextSeq 500
Strategy: ChIP-Seq
Source: OTHER
Selection: ChIP
Layout: PAIRED
Construction protocol: Following a shift at 37°C (duration depends on the mutant), after reaching exponential phase at 25°C in YPD, cells were cross-linked with 1% formaldehyde during 10 minutes. Cells were grown exponentially to 0.6 OD600 and cross-linked with 1% formaldehyde for 10 min. They were then washed with ST buffer (10 mM Tris-Cl pH 7.5, 100 mM NaCl) and frozen. Cells were lysed by bead-beating in FA lysis buffer (50 mM Hepes-KOH pH 8, 150 mM NaCl, 0.5 mM EDTA, 1% Triton, 0.1% sodium deoxycholate) supplemented with PMSF for 30 minutes at 4°C. Chromatin was recovered after a centrifuge step (16,000 rcf, 10 min, 4°C) in MNase buffer (20 mM Tris-HCl pH 7.5, 0.34 M sucrose, 15 mM KCl, 60 mM NaCl, 3 mM CaCl2) and was subjected to mild MNase (NEB, M0247S) treatment (20 min, 37°C on thermomixer). Reaction was stopped by addition of 10 mM EDTA on ice. Digested chromatin was recovered after centrifugation (16,000 rcf, 10 min, 4°C), and the remaining pellet was mildly sonicated (20” ON + 40” OFF, 4 cycles, mild), after a centrifugation step under similar conditions the chromatin samples were recovered at -80°C. The sizes of the DNA fragments have been checked by migrating the samples on both hand-made agarose and BioAanalyzer (Agilent) after pronase treatment. DNA was extracted with PCR purification kit (Qiagen). DNA was extracted using the Auto iPure V2 kit from Diagenode, following the manufacturer recommendations. Library construction was done according to the takara SMARTER thruplex (takara bio) protocol, following manufacturer's recommendation. Libraries were sized with AMPure beads (Beckman-coulter) to a final size of 325bp.