title: GSM4712693: BrdU_Input_G0_85_combined; Saccharomyces cerevisiae; ChIP-Seq
library_construction_protocol: For BrdU IP-seq, genomic DNA was isolated from each pulse-labelled sample by the “smash and grab” protocol (Hoffman, 1987), and 0.2 mg/ml RNase A was added for 30 min at 37C, followed by addition of 0.1 mg/ml of Proteinase K for 30 min at 50C.  DNA was purified with a Qiagen QIAquick PCR purification kit and its concentration was measured by an Invitrogen Qubit 2.0 Fluorometer.  DNA was transferred to a 50 l Covaris microTUBE and sheared to 200 – 250 bp using a Covaris M220 Focused-ultrasonicator (peak power: 75W; duty factor: 10%; cycles per burst: 200; treatment time: 400 sec). For Mcm4 ChIP-seq, twenty-four OD600 units of cells were collected and fixed with 1% formaldehyde for 15 min at room temperature.  Fixation was stopped by the addition of glycine to a final concentration of 125 mM for 5 min and the cells were washed with 50 mM Tris, pH 7.4, followed by centrifugation. Cell pellets were resuspended in 0.6 ml of ice-cold FA lysis buffer (1% Triton X-100, 0.1% sodium deoxycholate, 50 mM HEPES-KOH, pH 7.5, 140 mM NaCl, 1 mM EDTA, pH 8.0) supplemented with 1x protease inhibitor cocktail (Millipore-Sigma) and 1 mM of phenylmethylsulfonyl fluoride (PMSF), and then vortexed at highest speed with an equal volume of acid-washed glass beads (Millipore-Sigma) for 25 min at 4°C.  Cell lysates were sonicated using a Branson 250 Sonifier to generate DNA fragments of 200-500 bp.  After centrifugation for 15 min at 17,900 x g, soluble chromatin was collected from the supernatant fraction and protein concentration was determined by a Bradford assay.  Between 1-1.5 mg of chromatin was diluted in 1 ml of FA lysis buffer, and 20 µl was set aside as Input.  Anti-Myc (Millipore-Sigma) or anti-HA (Biolegend) antibodies were added to the remaining chromatin solution and incubation was continued overnight at 4°C  Protein G agarose beads (Invitrogen) were added for 2 h at 4°C, and the beads were then sequentially washed with FA lysis buffer, wash buffer I (1% Triton X-100, 0.1% sodium deoxycholate, 50 mM HEPES-KOH, pH 7.5, 500 mM NaCl, 1 mM EDTA, pH 8.0), wash buffer II (0.5% sodium deoxycholate, 0.5% NP-40, 10 mM Tris, pH 8.0, 250 mM LiCl), and TE, pH 8.0. Immunocomplexes were eluted from beads by incubation with 0.2 ml of elution buffer (1% SDS in TE) for 20 min at 65°C. Immunoprecipitated and Input samples were incubated with pronase solution (2 mg/ml pronase and 10 mM CaCl2) for 2 h at 42°C, followed by reverse crosslinking for 14-16 h at 65°C. DNA was purified with a Qiagen QIAquick PCR purification kit. For BrdU IP-seq, extracted genomic DNA was transferred to a 50 ml Covaris microTUBE and sheared to 200 – 250 bp using a Covaris M220 Focused-ultrasonicator (peak power: 75W; duty factor: 10%; cycles per burst: 200; treatment time: 400 sec).  One microgram of sheared DNA was end-repaired and ligated to Ion Torrent-compatible barcode adapters (Ion XpressTM Barcode Adapters, Invitrogen) using an Ion Xpress Plus Fragment Library kit (Invitrogen). The adapter-ligated DNA was purified by Agencourt AMPure XP Reagent and DNA concentration was measured by Qubit.  An equal concentration of each pulse-labeled G1 or G0 released DNA sample was pooled for immunoprecipitation (IP) and 20 ng of the mixture was set aside as input (IN) DNA. The pooled DNA was heated for 10 min at 95°C, followed by a snap cooling on ice.  Immunoprecipitation was performed with a 1:250 dilution of anti-BrdU antibody (GE Healthcare, RPN202) for 2h at 4°C, followed by the chromatin immunoprecipitation procedure described above. For Mcm4 ChIP-seq, multiple immunoprecipitation reactions were carried out in order to obtain a total of ~5 mg of immunoprecipitated DNA per sample.  For each sample, 5 mg of DNA was extracted from lysates and processed in parallel as input DNA.  IP and Input DNAs were processed for library preparation, including DNA end-repairing, barcoded adapter ligation, and PCR amplification using Ion Xpress Plus Fragment Library kit (Invitrogen).
attributes.tag: GEO Accession
attributes.value: GSM4712693