alias: GSM2809825
title: GSM2809825: pTpA_1E8_YPD_input_B2; Saccharomyces cerevisiae; MNase-Seq
library_construction_protocol: For each sample, 0.5mL of culture was pelleted and frozen to prepare input genomic DNA, and 3 mL of culture was crosslinked with 1% formaldehyde, washed twice with 1mL H2O supplemented with protease inhibitors cocktail, and the pellet frozen for MNase treatment. The MNase pellets were sphaeroplasted using zymolyase, and spheroplasts lysed in NP buffer (10 mM Tris pH 7.4, 50 mM NaCl, 5 mM MgCl2, 1 mM CaCl2, and 0.075% NP-40, freshly supplemented with 1 mM β-mercaptoethanol, 500 μM spermidine, and EDTA-free protease inhibitor cocktail) at a concentration of 2*10^6 cells/ul of NP buffer. 0.125 units of Worthington MNase were added per 10 ul of lysed spheroplasts and MNase digestion was preformed at 37C for 20 minutes. MNase digestion was stopped by addition of equal volume of 2X MNase Stop Buffer (220 mM NaCl, 0.2% SDS, 0.2% sodium deoxycholate, 10 mM EDTA, 2%,Triton X-100, EDTA-free protease inhibitor cocktail). MNased chromatin samples were treated with RNase A, and proteinase K, reverse cross linked, separated on a 4% agarose gel and mononucleosome bands were isolated. Genomic DNA was prepared using the Masterpure yeast genomic DNA preparation kit (Epicenter). For both MNase and genomic DNA, the variable region of the promoter library was amplified, and adaptors added for sequencing.
identifiers.namespace: GEO
identifiers.id: GSM2809825