title: GSM1143090: [MNase-Seq] Young yeast nucleosome replicate2; Saccharomyces cerevisiae; MNase-Seq
library_construction_protocol: The strains were grown in medium at 30°C overnight. The same number of young, old and histone H3/H4 overexpressing cells were collected and the cells were cross-linked with formaldehyde.  The cells were washed twice in 1xPBS buffer and pelleted. Then cells were resuspended in the Zymolyase buffer (1M sorbitol, 50 mM Tris-HCl at pH 7.4, and 10 mM b- Mercaptoethanol) for cell wall digestion with zymolyase-100T. After zymolyase-100T treatment, spheroplasts were pelleted and then resuspended in NP buffer (1M sorbitol, 50 mM NaCl, 10 mM Tris-HCl at pH 7.4, 5 mM MgCl2, 1mM CaCl2 and 0.075% Nonidet P40, and 500 mM spermidine). Micrococcal nuclease (MNase) was added to a final concentration of 1 U MNase/sample. The digestion reactions were incubated at 37°C for 30 min, and were stopped by adding SDS to a final concentration of 1% and EDTA to a final concentration of 10 mM. 5 ml of proteinase K solution was added to each tube. To reverse the formaldehyde cross-links, samples were incubated at 65°C overnight. Samples were extracted with 1 volume phenol/chloroform-IAA and centrifuged at room temperature for 15 min. DNA was precipitated with 2-propanol and washed with 70% ethanol. The pellet was dried at room temperature. 10 ml sterile ddH2O was added to the pellet. Then the DNA was treated with 1 ml RNase A (1mg/ml) at 37°C for 30 min. To check the mononucleosomal DNA fragments and MNase digestion level, electrophoresis was carried out on a 2% agarose gel. Samples were further analyzed on a Bioanalyzer 2100 to determine quality prior to library preparation. The libraries were generated using a KAPA library preparation kit (KK8200, Illumina Series).  A spike-in control which mimics the size of the mononucleosomal fragment was included during the library construction, and was added according to the number of cells used to isolate the mononucleosomal fragments.  The spike-in control used here comprises a set of three DNA sequences that were each around 150 nucleotides in length, with GC contents that mimic the GC% of the S. cerevisiae genome but with no significant similarity to the yeast genome. DNA fragments were size-selected between 200-400 bp after ligation of 121 bp adapters (Next flexTM DNA Barcodes-6).
attributes.tag: GEO Accession
attributes.value: GSM1143090