GSM9228274: WT_MNASE_T3_Rep1; Saccharomyces cerevisiae; MNase-Seq
For MNase-seq, 50 ml of synchronized meiotic cultures was harvested at the 3-hour time point and crosslinked with 1% formaldehyde for 30 minutes at room temperature. Crosslinking was quenched with 125 mM glycine, and cells were spheroplasted in Buffer Z (0.5 M sorbitol, 50 mM Tris-HCl pH 7.4, 10 mM β-mercaptoethanol) containing zymolyase. Spheroplasts were pelleted and resuspended in NP buffer (1 M sorbitol, 50 mM NaCl, 10 mM Tris-HCl pH 7.4, 5 mM MgCl₂, 1 mM CaCl₂, 0.5 mM spermidine, 1 mM β-mercaptoethanol, 0.075% NP-40) and digested with 5–80 units of micrococcal nuclease (MNase; Thermo Fisher Scientific, EN0181). MNase was diluted from a 300 U/µl stock to 20 U/µl, and mononucleosome-sized fragments were consistently obtained using 2 µl of a 1:4 dilution of this intermediate stock (10 units). Digestions were stopped with SDS and EDTA, followed by proteinase K treatment and overnight incubation at 65°C. Library Protocol:_x000B_ MNAse samples were processed as described in Blitzblau et al., 2012. The quality of the libraries was assessed using the 2100 Bioanalyzer or 2200 Tapestation. Library concentrations were quantified using qPCR prior to pooling. Sequencing was performed on Illumina NextSeq 500 instruments at the NYU Biology Genomics Core.