GSM6068835: spt650-2-HU-IP; Saccharomyces cerevisiae; OTHER
Cell pellets were resuspended in lysis buffer (2% SDS, 2% Triton-X100, 100 mM NaCl, 10 mM Tris pH 8.0, 1 mM EDTA) and 500 µL acid washed glass beads were added. Cells were lysed by vortexing for a total of 30 minutes at 4°C. Genomic DNA was isolated by phenol-chloroform isolation followed by ethanol precipitation with NaCl salt. DNA pellet was washed once with 70% ethanol and then resuspended in sterile water. DNA was treated with RNaseA/T1 for 30 minutes at 37°C followed by Proteinase K for 30 minutes at 50°C. Genomic DNA (gDNA) was column purified by a PCR purification kit. gDNA was fragmented by sonication to ~300 bp. Sonication efficiency was confirmed by gel electrophoresis. DNA concentration was measured by nanodrop and an equal amount of sonicated S. pombe genomic DNA was added to each sample. Input samples were removed immediately following spike-in addition. For next-generation sequencing experiments, libraries were prepared with the Qiagen GeneRead kit according to manufacturer's instructions prior to IP. For the IP, gDNA was boiled for 5 minutes at 95°C. Samples were incubated on ice for 3 minutes. 50 µL of gDNA (50-500 ng in EB) was combined with 50 µL anti-BrdU (1:250 in 2X IP buffer (2XPBS, 0.001% Triton-X100)) and incubated on a rotisserie for 2 hours at 4°C. 15 µL of washed Protein G Dynabeads was added. Samples were incubated on a rotisserie for 1 hour at 4°C. Samples were washed thrice in 1XIP buffer. Sample was eluted by the addition of TES and incubating for 10 min at 65°C. Libraries were prepared with Qiagen GeneRead Kit according to manufacturers instructions.