title: GSM3756713: H4ac Nrd1-AA_-Rap_rep1; Saccharomyces cerevisiae; ChIP-Seq
library_construction_protocol: The MNase- and MNase-ChIP-seq experiments were performed mainly as described in the (Weiner et al., 2015) with the following modifications. Nrd1-AA strain was inoculated overnight and diluted in the morning to OD600 of 0.2 in YEPD medium. Yeast cell culture of 100 mL was used per modification. Rapamycin treatment was performed at OD600 of 0.5 for 60 minutes on half of the culture. The cells were fixed with formaldehyde at a final concentration of 1% for 15 minutes followed glycine addition at a final concentration of 125 mM for 5 minutes. Cells were washed 2x with ddH2O , harvested in magnaLyser tubes (Roche) (100 mL cell culture/tube) and frozen at -20 °C .   Chromatin extraction was performed by breaking cells in 1 mL of Cell Breaking buffer (0.1 M Tris-HCl, pH 7.9, 20% glycerol, EDTA-free protease inhibitors) and 1 mL of acid-washed glass beads in a magnaLyser (Roche). The cell extract was then centrifuged at 19,000 g at 4 °C for 10 minutes to collect chromatin. The chromatin was resuspended in 600 uL of NP buffer (0.5 mM Spermidine, 50 mM NaCl, 1mM beta-mercaptoethanol, 0.075% NP-40, 10 mM Tris-HCl pH 7.4, 5 mM MgCl2, 1 mM CaCl2) per tube. The DNA concentration was measured using Qubit dsDNA BR assay kit (Invitrogen). Chromatin was diluted to 20 ug/mL using NP buffer in all conditions for comparable results.   MNase treatment was performed using 0.2 uL of MNase (ThermoScientific 100 units/uL ) / 12 ug of chromatin in 600 uL reaction mixture for 14 minutes at 37 °C. To perform all the modifications for one replicate from one cell culture the reaction was scaled 10x. The reaction was stopped by adding EDTA (final concentration 20 mM) in the reaction mixture.   For MNase-seq, equal amount of elution buffer (10 mM Tris-HCl, pH 8.0, 1% SDS, 150 mM NaCl, 5 mM DTT) was added. The further steps for MNase is same as the ones performed after MNase-ChIP elution.  For MNase-ChIP, the buffer of the MNase reaction mixture was adjusted to be compatible to FA-lysis buffer (50 mM HEPES-KOH, pH 7.5, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, EDTA-free protease tablet).  The following salts were added to the MNase reaction tube: 80 uL of 0.5 M HEPES-KOH, pH 7.5, 22.4 uL of 5 M NaCl,  pre-mixed with protease inhibitors. Followed by addition of detergents, 6.4 uL of 12.5% sodium deoxycholate and 80 uL of 10% Triton X-100 per 600 uL MNase reaction.   The protein-G dynabeads (ThermoFisher Scientific) were pre-incubated with the antibodies for one hour at 4 °C on a rotor, 85 uL beads/reaction. The antibodies used were anti-H3 (Abcam ab1791) 12 uL  per reaction, anti-H3K36me3 (Abcam ab9050) 6 uL  per reaction, anti-H3K18ac (abcam ab1191) 7 uL  per reaction, H4ac (Merck Millipore 06-598) 10 uL  per reaction. The beads were washed in FA-lysis buffer twice, followed by incubation with the MNase-treated chromatin for 4 hours at 4 °C on a rotor. The beads were sequentially washed with 1 mL of  FA-Lysis buffer, 1 mL FA500-lysis Buffer (FA-Lysis Buffer + 500 mM NaCl), 1 mL of Buffer III ( 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 250 mM LiCl, 0.5% NP-40, 0.05% sodium deoxycholate), 1 mL of Tris-EDTA pH 8.0. For elution, the beads were incubated in 150 uL of elution buffer at 65 °C for 15 minutes.   The crosslinking was reversed by incubation of the eluate at 65 °C overnight followed by addition of 150 uL of TE (10 mM Tris-HCl, pH 8.0, 1 mM EDTA). The eluate was then treated with proteinase K (1 ug/uL final concentration) for 3 hours at 42 °C. The DNA was purified using the NucleoSpin Gel and PCR clean-up kit (Macherey-Nagel) using the SDS protocol provided in the manual. The DNA concentration was measured using Qubit dsDNA HS assay kit (Invitrogen) and the libraries were prepared using NEBnext Ultra DNA library prep kit for Illumina (NEB). Finally, samples were paired-end sequenced at the iGE3 genomics platform of the University of Geneva. NEBnext Ultra DNA library prep kit for Illumina (NEB)
attributes.tag: GEO Accession
attributes.value: GSM3756713