title: GSM4491310: WT_MCM_ChIP-seq; Saccharomyces cerevisiae; ChIP-Seq
library_construction_protocol: For ORC and MCM ChIP, WT and MUT (orc4-IHΔ) yeast strains with Myc-tagged Orc6 and FLAG-tagged Mcm7 were arrested at G2/M and G1 phase respectively. Chromatin extracts were prepared and immunoprecipitated as previously described with minor modifications. About 2x109 cells were crosslinked by adding formaldehyde (Millipore) to a final concentration of 1% and incubated at room temperature for 25 minutes with gentle shaking. Cross-linking reaction was halted by adding glycine to a final concentration of 0.125 M and incubation for additional 10 mins at room temperature. Cells   were lyzed by re-suspension in lysis buffer and subjected to bead-beating using a Mini-Beadbeater (Biospec). Chromatin fraction was collected by centrifugation and were sonicated to yield an average DNA size of 300bp using ultrasonicator (Covaris). Sonicated chromatin fractions were clarified by centrifugation and ~400 ml of clarified fraction was subjected to immunoprecipitation with 60 ml of protein G-Dynabeads (Invitrogen) conjugated to anti-Myc antibody (Roche, 9E10) for Orc6-Myc ChIP or anti-FLAG antibody (Sigma, M2) for Mcm7-FLAG ChIP at 4ºC overnight. Immunoprecipitated DNA was then recovered as previously described43. Reverse crosslinked samples were digested with RNase A (Roche) for 1 hour followed by Proteinase K (Roche) for two hours at 37ºC. DNA was purified using MinElute PCR purification column according to Qiagen protocol. Validation of the ChIP was carried out by qPCR targeting positive control loci using WT DNA.  Sequencing libraries for ChIP-seq, BrdU-IP-seq and MNase-seq were prepared using Kapa Hyper Prep Kit according to manufacturer's protocol.
attributes.tag: GEO Accession
attributes.value: GSM4491310