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| Status |
Public on Nov 27, 2019 |
| Title |
WT3_AluI_100U_30min |
| Sample type |
SRA |
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| Source name |
chromatin
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| Organism |
Saccharomyces cerevisiae |
| Characteristics |
restriction enzyme (re): AluI
strain: BY4741 - Replicate 3
units of re/incubation time: 100/ 30 min
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| Treatment protocol |
Yeast nuclei were prepared as described (Almer et al. 1986). In brief, cells were harvested by centrifugation, washed in water, resuspended in 2.8 mM EDTA, pH 8, 0.7 M 2-mercaptoethanol, incubated for 30 minutes at 30°C, washed in 1 M sorbitol, resuspended in 1 M sorbitol, 5 mM 2-mercaptoethanol and spheroplasted by incubation with 20 mg/ml freshly added Zymolyase 100T (MP Biochemicals) for 30 minutes at 30°C. Spheroplasts were washed with 1 M sorbitol and resuspended in lysis buffer (18% Ficoll, 20 mM KH2PO4, 1 mM MgCl2, 0.25 mM EGTA, 0.25 mM EDTA) followed by centrifugation at 22,550 g for 30 min to collect chromatin (“nuclei”). Pellets were frozen in dry-ice/ethanol and stored at -80°C.
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| Growth protocol |
The BY4741 strain (MATa his3D0 leu2D0 met15D0 ura3D0, Euroscarf) was grown to log phase (Supplemental Fig. S1A) in YPDA medium (1% w/v Bacto yeast extract, 2% w/v Bacto peptone, 2% w/v glucose, 0.1 g/l adenine, 1 g/l KH2PO4).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Nuclei from ca. 0.1 g wet cell pellet were pre-washed in methylation buffer, centrifuged and resuspended either in 400 µl 1x CutSmart buffer (NEB) for BamHI-HF and AluI or in 1x NEB2.1 for HindIII. Digestions were started by RE addition, incubated for 30 or 120 minutes at 37°C and stopped by 10 mM EDTA and 0.5% SDS (final conc.). DNA preparation was as above. For the cut-all cut method, Schizosaccharomyces pombe gDNA, fully digested with the corresponding RE, was spiked in at 5-10% DNA mass of the final sample before phenol-chloroform extraction. After RNase A digestion, half of the sample was digested for a second time with 100 U of the corresponding RE and in the corresponding buffer for 1.5 h at 37°C. The reaction was stopped by 15 mM EDTA (final conc.).
Purified DNA was sheared to ~150 bp fragments with the Covaris S220 and concentrated using the NucleoSpin Gel and PCR Clean-up Kit (Macherey&Nagel). 0.5 – 1 µg as determined by Qubit (ThermoFisher Scientific) was used for library preparation. First, DNA was end polished (15 U T4 DNA Polymerase, 50 U T4 PNK, 5 U Klenow (NEB)) for 30 minutes at 20°C, purified with AmPure XP beads (Beckman Coulter), then subjected to A-tailing (15 U Klenow exo-(NEB)) for 30 minutes at 37°C and purified again. For adapter ligation 15 U T4 DNA Ligase (NEB) and 75 to 150 pmol NEBNext Adaptor or NEBNext Methylated Adaptor for bisulfite conversion were added. The reaction was incubated for 20 minutes at 25°C followed again by AmPure XP bead purification. Half of the end-polished and adapter ligated sample was subjected to 8 cycles of PCR using the NEBNext Multiplex Primers for Illumina and Phusion Polymerase (NEB). Methylated samples were bisulfite converted following the manufacturer’s instructions of the Qiagen EpiTect Bisulfite Kit. 3 µl of the converted sample was subjected to 12-14 cycles of PCR using the NEBNext Multiplex Primers for Illumina and Phusion U Polymerase (ThermoFisher Scientific). All samples were sequenced by LaFuGa (Illumina HiSeq 1500, 50 bp paired-end mode).
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 1500 |
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| Data processing |
Library strategy: ORE-seq
ORE-seq: cut and uncut fragment count and resection length After demultiplexing (Girardot et al. 2016), sequenced reads were trimmed based on basecalling quality and mapped (Li and Durbin 2009) using the combined S. cerevisiae and S. pombe reference genome. Fragments longer than 500 bp or on rDNA loci were removed. At each cut site, fragment starts and ends were counted within a sample-specific window (green areas in Fig. S1D, see Supplemental Methods) to account for exonuclease resection and within the same window the mean resection length was calculated. Uncut fragments, i.e. fragments covering, but not starting or ending at the cut site, were also counted at each cut site. Cut sites with neighbours closer than 200 bp were ignored completely and remaining cut sites with neighbours closer than 300 bp were analysed only using the starting/ending counts not pointing towards the close neighbour. ORE-seq: occupancy estimation (cut-uncut method) See Methods and Supplemental Methods.
Calculation of ORE-seq and ODM-seq maps For RE samples, very rare occupancy estimates outside of the interval between 0 and 1 were truncated to 0 or 1. For the ORE-seq map (Fig. 1B) we averaged the occupancy values at the same sites in different RE samples with equal weights. Similarly, for the ODM-seq map (Fig. 1B), different bisulfite samples were averaged with equal weights. We also calculated individual enzyme maps in the same way (attached as supplementary file).
BedGraph files contain the (corrected) absolute occupancy values and were generated using the R (R_Core_Team 2018) package rtracklayer (Lawrence et al. 2013).
tsv files contain columns with the absolute occupancy (corrected and not corrected), the number of effective cuts and the effective coverage for each RE site.
Genome_build: SacCer3
Supplementary_files_format_and_content: bedGraph, tsv
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| Submission date |
Nov 26, 2019 |
| Last update date |
Nov 27, 2019 |
| Contact name |
Elisa Oberbeckmann |
| E-mail(s) |
elisa.oberbeckmann@med.uni-goettingen.de
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| Phone |
+49 551 39 65975
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| Organization name |
University Medical Center Göttingen
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| Department |
Molecular Biology
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| Lab |
Oberbeckmann
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| Street address |
Justus-von-Liebig Weg 11
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| City |
Göttingen |
| ZIP/Postal code |
37077 |
| Country |
Germany |
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| Platform ID |
GPL18085 |
| Series (2) |
| GSE132225 |
Absolute nucleosome occupancy map for the Saccharomyces cerevisiae genome |
| GSE141056 |
Absolute nucleosome occupancy map for the Saccharomyces cerevisiae genome [ORE-seq] |
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| Relations |
| BioSample |
SAMN13390353 |
| SRA |
SRX7218416 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4193303_WT3_AluI_100U_30min.bedgraph.gz |
83.1 Kb |
(ftp)(http) |
BEDGRAPH |
| GSM4193303_WT3_AluI_100U_30min.tsv.gz |
178.8 Kb |
(ftp)(http) |
TSV |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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