GSM1964539: Pro; Saccharomyces cerevisiae; MNase-Seq
Yeast were treated with 2% formaldehyde for 30 minutes (identical results are obtained with 15 minute incubations), stopped with 125 mM glycine,  then collected by centrifugation, washed, and treated with zymolyase (Seigaku) at 0.25 mg/ml. Then the Lysates were digested with micrococcal nuclease, treated with proteinase K and reverse formaldehyde crosslinks. Finally the nucleosome DNA were phenol:chloroform extracted, ethanol precipitated, and treated with DNase-free RNase. Samples were examined by gel electrophoresis, and reactions with ~90% mononucleosomal DNA were used for squencing. Libraries were prepared according to Illumina's instructions accompanying the TruSeq® Nano DNA LT Sample Preparation Kit (Part# FC-121-4001). Briefly, DNA was end-repaired using a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (32 to 52 exo minus) and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters which have a single 'T' base overhang at the 3’ end. After adapter ligation DNA was PCR amplified with Illumina primers for 15 cycles and library fragments of ~250 bp (insert plus adaptor and PCR primer sequences) were band isolated from an agarose gel. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the Genome Analyzer following the manufacturer's protocols.
GEO Accession
GSM1964539