GSM3325426: pob3-Q308K H3-K56Q1; Saccharomyces cerevisiae; MNase-Seq
Cells were subjected to cross-linking with formaldehyde (1%) for 20 min at room temperature. Glycine (final 0.2 M) was added to quench crosslinking and cells were washed with 1X Tris-buffered saline. Cell walls were removed with 500 U lyticase, 37° 20 minutes, or until >90% of cells are spheroplasted and collected by centrifugation. Cells were lysed in MNase digestion buffer and 10 U MNase, then incubated at 37° for 15 minutes, and the reaction stopped, diluted, and crosslinks reversed by incubating 5 hours at 65°. DNA was precipitated with ethanol, then treated with RNase A, followed by Proteinase K digestion and purification on Qiagen Qiaquick columns. Sequencing libraries were constructed using NEBNext® Multiplex Oligos (Index Primers Set 1 and Set 2) and NEBNext® ChIP-seq Library Prep Reagent Set for Illumina. Ligated samples were purified by electrophoresis through 1.5% agarose, excising the 270 bp mononocleosome band (150 bp + 120 bp adaptors), purified on a Qiagen gel extraction kit. Samples were then subjected to 50 bp single-end sequencing in an Illumina Hiseq2500 sequencer.
GEO Accession
GSM3325426