7 protocols
AccessionType
high throughput sequence alignment protocol
The paired-end reads were aligned to the 2008 version of the Saccharomyces Cerevisiae genome (sacCer2) using Bowtie (version 0.12.7 (Langmead et al, 2009)) allowing for 2 mismatches in the first 40 bp of the read. More than 90% of the reads were mapped to the yeast genome.
normalization data transformation protocol
Based on previous work (Zhou and O'Shea, 2011) we restricted our analysis to reads of between 100 and 200 bp and equated the center of the read to the nucleosome dyad. We thus aligned the center of each sequenced nucleosomal DNA fragment on the genome and extended 73 bp on both sides to generate mono-nucleosome coverage (using MATLAB). The occupancy score was subsequently normalized. Our nucleosome maps closely match previous maps (Zhou & O'Shea, 2011) despite differences in culturing conditions, strain background etc. and were highly correlated with each other (Pearson's correlation coefficient generally: p 0.95). The data files iRFP_Nuc_3uM_0min.txt, ..., iRFP_Nuc_3uM_40min.txt contain the nuclesomes aligned to the SacCer2 Cerevisiae reference genome. The first column in these files gives the chromosome number (e.g. chr2), the second column gives the position of the nucleosome centre (dyad) on that chromosome (position 82017 on chr3) and the third and last column gives the length of the paired-end sequencing read.
nucleic acid sequencing protocol
Sequencing libraries were sequenced from both ends on an Illumina HiSeq 2000 machine generating 179 million paired-end reads of 100 bp each. We obtained between 24 and 32 million paired-end reads for each timepoint.
nucleic acid library construction protocol
Sequencing libraries were prepared following the Illumina Multiplexed paired-end protocol. Adapters with a different index (0 min: ATCACG, 5 min: CGATGT, 10 min: TTAGGC, 20 min: TGACCA, 30 min: ACAGTG and 40 min: GCCAAT) for each timepoint were ligated onto the nucleosomal DNA fragments and the sequencing library amplified by 8 cycles of PCR.
nucleic acid extraction protocol
The frozen cell pellet was thawed and 200 uL lysis buffer was added (50 mM HEPES, pH 7.5, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1% Sodium deoxycholate, complete protease inhibitor cocktail tablets (Roche cComplete Mini version 10, 1 tablet per 10 mL buffer)). The cells were then mechanically lysed with glass beads and bead beating. The cell pellets were then washed twice with MNase reaction buffer (10 mM Tris pH 7.5, 50 mM NaCl, 5 mM MgCl2, 1 mM CaCl2, 1 mM bME), resuspended in 750 uL MNase buffer and digested to primarily mononuclesomes with 0.75-1.5 U of MNase at 37 degrees C for 30 minutes. Subsequently samples were quenched by addition of 15 uL EDTA (500 mM). Debris was removed by centrifugation and 16 uL SDS (20%) and 30 uL proteinase K (10 mg/mL) were added to the supernatant. Crosslinks were then reversed and proteins digested by incubation at 65 degrees C overnight. The DNA was then purified twice by phenol/chloroform/isoamy alcohol extraction (25:24:1) and followed by precipitation in 1 mL ethanol at -20 degrees C. Precipitated DNA was resuspended in RNase A (0.2 mg/mL in TE buffer) and residual RNA removed by digesting for 1 hour at 37 degrees C. The samples were then purified using the PCR Purification Kit (Qiagen) and run on a 1.5% agarose gel for 60 minutes. The mononucleosome bands at ca. 150 bp were excised and purified on a Freeze 'N Squeeze DNA gel extraction column (BioRad). The DNA was then collected by ethanol precipitation.
treatment protocol
The experimental protocol was essentially identical to what was previously described (Zhou & O'Shea, 2011). Briefly, 6 L of diploid (ASH79) was grown overnight in low fluorescence medium (-TRP -LEU) and then split into six 1L cultures. 40, 30, 20, 10, 5 and 0 min before the cultures reached an OD600 nm of 0.150 1-NM-PP1 was added to a final concentration of 3 uM. Cells were then crosslinked with 1% formaldehyde (final concentration) for 15 minutes at room temperature and quenched for 5 minutes with 125 mM glycine (final concentration). Cells were collected by centrifugation and immediately washed (10 mM Tris pH 7.5, 100 mM NaCl). The cell pellet was then dried and snap frozen in liquid nitrogen.
growth protocol
The experimental protocol was essentially identical to what was previously described (Zhou & O'Shea, 2011). Briefly, 6 L of diploid (ASH79) was grown overnight in low fluorescence medium (-TRP -LEU) and then split into six 1L cultures. 40, 30, 20, 10, 5 and 0 min before the cultures reached an OD600 nm of 0.150 1-NM-PP1 was added to a final concentration of 3 uM. Cells were then crosslinked with 1% formaldehyde (final concentration) for 15 minutes at room temperature and quenched for 5 minutes with 125 mM glycine (final concentration). Cells were collected by centrifugation and immediately washed (10 mM Tris pH 7.5, 100 mM NaCl). The cell pellet was then dried and snap frozen in liquid nitrogen.