title: GSM3332093: MNase-seq_G2; Saccharomyces cerevisiae; MNase-Seq
library_construction_protocol: For MNase digestions 160 ml of cells (OD600=1) where fixed with 1 % formaldehyde for 30 minutes at 25 °C with 100 r.p.m. agitation and quenched with 125 mM glycine for 10 minutes at 25 °C with 100 r.p.m. agitation. Cells were spun and washed twice with PBS. Pellet was resuspended in 20 ml sorbitol/ tris buffer (1M sorbitol, 50 mM Tris-HCl pH 7.5, 10 mM β-mercaptoethanol) containing 10 mg of Zymolyase 20T and incubated at 30 °C for 5 minutes or until spheroplast were formed. Spheroplasts were spun at 3,000 r.p.m. for 5 minutes and washed with sorbitol/tris buffer without b-mercaptoethanol. Spheroplasts were resuspended in NP buffer (1 M sorbitol, 50 mM NaCl, 10mM Tris pH 7.5, 5 mM MgCl2, 1mM CaCl2, 1mM b-mercaptoethanol, 500 μM spermidine, 0.075 % NP40) and digested with 600, 1200 or 1800 U/ ml of MNase (NEB) for 20 minutes at 37 °C. MNase digestion was stopped adding 200 μl of STOP solution ( 5% SDS, 50 mM EDTA). DNA was decrosslinked by adding 1 mg/ml proteinase K and incubating at 65 °C overnight. DNA was purified by adding 0.6M potassium acetate pH 5.5 and incubating on ice 5 minutes and centrifuging 10 minutes at 3,500 r.p.m followed by a phenol chloroform extraction. Finally DNA was precipitated by adding 25 μg/ml glycogen, 1/25 columes NaCl, 0.7 volumes Isopropanol, incubating 30 minutes at -20 °C and centrifuging 30 minutes at 13,200 r.p.m. The pellet was washed in 70 % ethanol and resuspended in TE 1X. Samples were run in parallel in a 1.5 % agarose gel and the mononucleosomes of a digestion with a ratio of 80:20 mononucleosomes to dinucleosomes were purified and used in Next Generation Sequencing Libraries of mononucleosomal and ChIP DNA were constructed following the Illumina compatible NEXTflexTM ChIP-Seq kit protocol and sequenced in an Illumina NextSeq500 platform using the paired-end protocol. Reads were aligned to the S.cerevisiae (SacCer3) genome using Bowtie. Nucleosome occupancy maps were generated using the NUCwave algorithm.  Nucleosome fuzziness was analyzed using the dpos utility of DANPOS2 application. To make it compatible with NUCwave maps, we used the following parameters: a span of 1 bp and a read extension of 50 bp. ChIP-seq data representation was performed using the Model-based Analysis of ChIP-Seq (MACS) with a read extension of 147 bp.