title: GSM1891208: RBP1_02; Saccharomyces cerevisiae; MNase-Seq
library_construction_protocol: Chromatin for MNase digestion was prepared essentially as described (Kent and Mellor, 1995). Yeast cultures were grown at 30°C in YPAD to OD600~0.4 and crosslinked with 1% formaldehyde for 5 minutes followed by 5 minutes of quenching with 125 mM glycine. After wash with 1M sorbitol, spheroplasts were derived from the cultures by 8 minute treatment with 10 mg/ml Zymolase (100T, USBiological) in spheroplasting buffer (1 mM β-mercaptoethanol, 1 M sorbitol) at room temperature. Next the spheroplasts were washed twice with 1 M sorbitol without disrupting the pellet and treated with various concentrations of MNase (Sigma), ranging from 0.2 to 4 units in digestion buffer (1 M sorbitol, 50 mM NaCl, 10 mM Tris [pH 7.4], 5 mM MgCl2, 1 mM CaCl2, 1 mM β-mercaptoethanol, 0.5 mM spermidine, 0.075% NP-40). Spheroplasts derived from an equivalent of 50 ml of culture served as one sample. The samples were incubated at 37°C for 45 minutes. The reactions were stopped by addition of EDTA (30 mM final conc.) and the crosslinks were reversed by addition of SDS (0.5% final conc.) and proteinase K (0.5 mg/ml final conc.), incubation at 37°C for 1h followed by incubation at 65°C for at least 2h. Samples underwent phenol-chloroform extraction and DNA was precipitated from the aqueous phase using ammonium acetate and ethanol. After wash with 70% ethanol, the DNA pellet was dried, resuspended in 30 μl TE with RNase (0.1 mg/ml final concentration) and the samples were incubated for 30 min at 37°C to digest RNA. 2 μl aliquots were resolved on 3% agarose gel to evaluate the digestion. The samples from anchor-away experiments treated with vehicle and rapamycin chosen for deep sequencing that were to be compared were digested to exactly the same extent, as evaluated by the intensity ratios between mono- and di-nucleosomal DNA bands. TruSeq paired-end (Illumina).
identifiers.namespace: GEO
identifiers.id: GSM1891208