title: GSM4849503: Untagged_100u_Mnase-Input; Saccharomyces cerevisiae; ChIP-Seq
library_construction_protocol: MNase ChIP-seq: cell pellets were resuspended in lysis buffer (50mM HEPES pH 7.5, 140mM NaCl, 1mM EDTA, 2% Tritox X-100, 0.2% Na-deoxycholate, 1X roche protease inhibitor cocktail, 1mM PMSF) and bead beat for 25 minutes. Cell lysates were spun down at 15,000g for 30 minutes. The chromatin pellets were re-suspended in 1000 μl of NP-S buffer (0.5 mM spermidine, 1 mM β-ME, 0.075% NP-40, 50 mM NaCl, 10 mM Tris pH 7.4, 5 mM MgCl2, 1 mM CaCl2) and digested with 100u microccocal nuclease at 37°C for 10 minutes to obtain predominantly mono-nucleosomal DNA. Reactions were stopped with the addition of 10 mM EDTA and digested lysates were clarified by centrifugation at 9000g for 10 minutes. To extract insoluble chromatin, pellets were re-suspended in 300 μl of lysis buffer with 0.2% SDS, and sonicated in a Diagenode Bioruptor at high output for 30 seconds on/30 seconds off four cycles. Extracts were then re-clarified by centrifugation at 9000g for 10 minutes, and the supernatant pooled with the pre-existing extract. The buffer composition of the lysate was adjusted to that of the original lysate buffer (50mM HEPES pH 7.5, 140mM NaCl, 1mM EDTA, 2% Tritox X-100, 0.2% Na-deoxycholate, 1X roche protease inhibitor cocktail, 1mM PMSF), and 10% was set aside as input. The supernatant was pre-cleared by incubation with magnetic protein-G Dynabeads (Life Technologies) for 1 hour at 4°C. Pre-cleared lysates were incubated with antibodies at 4°C overnight. Immune complexes were precipitated by incubation with magnetic protein-G Dynabeads for one hour at 4°C. Beads were washed twice with lysis buffer, high salt wash buffer (50mM HEPES pH 7.5, 640mM NaCl, 1mM EDTA, 2% Tritox X-100, 0.2% Na-deoxycholate), LiCl was buffer (10mM Tris-HCL pH 8.0, 250mM LiCl, 0.6% NP-40, 0.5% Na-deoxycholate, 1mM EDTA), and once with TE.  Sequencing libraries were prepared as described previously (Maltby et al. 2012; Martin et al. 2017). Briefly, 2 ng of ChIP or input material was end-repaired, A-tailed, and adapters ligated, before PCR amplification with indexed primers. We performed 10 and 11 rounds of PCR amplification for MNase and sonication ChIP-seq experiments, respectively. All DNA purification steps used SPRI (Solid Phase Reversible Immobilization) magnetic beads. Samples were pooled, gel-purified (50 bp - 600 bp region) and sequenced on an Illumina HiSeq 2500.
attributes.tag: GEO Accession
attributes.value: GSM4849503