title: GSM2462584: Rpo21 glucose to no glucose anchor away strain rapamycin time point 14; Saccharomyces cerevisiae; OTHER
library_construction_protocol: Cells were lysed in 1V/w of TN150 (50 mM Tris pH 8.0, 150 mM NaCl, 0.1% NP-40, 5 mM b-mercaptoethanol) and 3V of Zirkonia beads (0.5 mm; Thistle Scientific) as previously described (Granneman et al. PNAS 2009). For the Nab3 anchor-away nuclear depletion experiments (Haruki et al., Mol Cell 2008), cells were incubated with 1µg/ml rapamycin (Sigma) for 1 hour before the cells were shifted.  For the Pol II (Rpo21-HTP) cCRAC experiments, cells were lysed in 1V/w TMn150 (50 mM Tris-HCl pH 8.0, 10 mM MnCl2, 0.1% NP-40, 5 mM b-mercaptoethanol, 150 mM NaCl, Roche Midi protease inhibitors; 1 ml per gram of cells). Subsequently, 1V/w of TMn150 was added containing 1U/ml of RQ1 RNase-free DNase (Promega) and the suspension was incubated for half an hour on ice to degrade the chromatin. Three milliliter of lysis buffer was added and extracts were clarified by centrifugation (20 min at 4,500 g and 20 min at 20,000 g at 4 °C). Extracts were incubated with 250 µl of equilibrated IgG Sepharose beads (GE Healthcare) for 2h at 4 °C. linker ligations were performed as previously described (Granneman et al., PNAS 2009) using adapters provided in the Supplementary Information of the manuscript. Cross-linked protein-RNA complexes were resolved on 1mm thick 4-12% NuPAGE gels (Thermo Fisher Scientific), transferred to nitrocellulose membranes and visualized by autoradiography. Bands corresponding to the size of the protein of interest, including a region 0.5 cm above the band, were cut from the nitrocellulose membrane and pooled in a single 2 ml tube. Radiolabeled RNA was extracted by incubating the membrane slices with 200 µg of proteinase K in 800 µl of wash buffer II containing 1% SDS and 5 mM EDTA. The solution was transferred to a new tube and the RNA was subsequently phenol-chloroform-extracted and ethanol-precipitated. Reverse transcription with SuperScript III was performed as per manufacturer's procedures (Thermo Fisher Scientific) using the reverse transcription primer listed in Table S2. The cDNAs were purified using the Zymo DNA Clean & Concentrator 5 kit and eluted into a final volume of 10 µl. Five µl of cDNA was PCR amplified using Pfu polymerase (Promega) for 20-24 cycles (95ºC 30s, 52ºC 30s and 72ºC 1min) using PCR oligonucleotides listed in Table S2. PCR products were resolved on 2% Metaphor agarose gels (Lonza) and 160-300 bp fragments were gel purified using the miniElute kit (Qiagen) according to manufacturer’s procedures. Paired-end sequencing (50 bp) was performed by Edinburgh Genomics using the Illumina HiSeq 2500 and 4000 platforms.
attributes.tag: GEO Accession
attributes.value: GSM2462584