GSM8640710: CRAC-seq of Mmyco strain, rep2; Saccharomyces cerevisiae; OTHER
Cells were exposed to UV light using a W5 UV crosslinking unit (UVO3 Ltd) for 50 seconds, harvested and resuspended in TN150 buffer (50 mM Tris pH 7.8, 150 mM NaCl, 0.1% NP-40 and 5 mM beta mercaptoethanol, 2.4 ml/g of cells) with protease inhibitors. Cells were broken using a Mixer Mill MM 400. Extracts were treated for one hour at 25°C with DNase I (165 U/g of cells) to solubilize chromatin and then clarified by centrifugation (20 min at 20000g at 4°C). Rpb1-RNA complexes were purified by a two-step procedure using the protein A and the his-6 portion of the tag, with TEV cleavage for eluting complexes at the proteinA-dependent purification step. Adaptors were added during the purification steps. The protein-RNA adduct was further purified by denaturing PAGE and the RNA recovered by proteinase K degradation and phenol extraction. The library was amplified by 10 cycles of PCR after reverse transcription and sequenced using Illumina technology