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| Status |
Public on Nov 27, 2019 |
| Title |
WT1_xl_GpC_200U_180min |
| Sample type |
SRA |
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| Source name |
chromatin
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| Organism |
Saccharomyces cerevisiae |
| Characteristics |
methyltransferase: GpC
strain: BY4741 crosslinked - Replicate 1
units of dnmt/incubation time: 200/ 180 min
chromatin stabilization: crosslinked cells
conversion method: BS-seq
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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). Crosslinking was with 1% formaldehyde (final conc.) for 1, 5 or 20 minutes at RT while shaking and quenched for 20 min with 125 (WT1, WT4) or 250 (WT5) mM glycine (final conc.).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Nuclei pellets were washed in methylation buffer (20 mM HEPES-NaOH pH 7.5, 70 mM NaCl, 0.25 mM EDTA pH 8.0, 0.5 mM EGTA pH 8.0, 0.5% (v/v) glycerol, 1 mM DTT, 0.25 mM PMSF (Darst et al. 2012)). Per reaction, nuclei from ca. 0.1 g wet cell pellet were resuspended in 800 µl methylation buffer containing 640 µM freshly added S-adenosylmethionine (SAM). 200 U M.SssI or M.CviPI (both NEB) and 10 mM DTT were freshly added. Methylation reactions were dialyzed in Slide-A-Lyzer MINI Dialysis Devices 10 K MWCO (Thermo Fisher Scientific) against 15 ml methylation buffer with 200 µM freshly added SAM at 25°C for M.SssI or 37°C for M.CviPI. 0.5 - 1 µg fully assembled pUC19-601-25mer plasmid (pFMP233) (Lowary and Widom 1998; Lieleg et al. 2015a) and SGD assembled Escherichia coli gDNA or SGD assembled E. coli plasmid library (limited Sau3A fragments of E. coli gDNA ligated into pJET 1.2 plasmid (Thermo Fisher Scientific)) was spiked-in prior to methylase addition. E. coli spike-in results were not further pursued due to low coverage. Reactions were stopped by 0.5% SDS (final conc.), reversed crosslinked if required and DNA was deproteinized by Proteinase K, phenol-chloroform extracted and RNase A digested.
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: ODM-seq
ODM-seq analysis for BS-seq and EM-seq data We mapped the paired-end reads with BS-Seeker2 (version 2.1.8, (Guo et al. 2013)) using Bowtie (Langmead et al. 2009) and trimmed the reads at the real fragment ends by 5 bp to 10 bp to obtain a constant conversion ratio along reads averaged over all reads, as the conversion ratio usually shows an increase or decrease at these ends due to end repair. We ignored reads on the loci of rDNA genes and reads with an unconverted HCH motif, as HCH should be fully converted. The “anti pattern” of the CpG/GpC DNA methyltransferases is the GCH/HCG pattern, respectively, and should also be fully converted. We discarded a sample, if the average anti pattern conversion ratio among all reads was less than 0.98. At each CpG/GpC methylation site the occupancy is calculated as ratio of the converted reads over the number of all reads. We ignored methylation sites with a coverage less than 20.
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 (maps attached as supplementary file). We also calculated individual enzyme maps in the same way.
BedGraph files contain the 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 value, the strand, the motif and the number of converted as well as the total number of reads for each methyation 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 |
| GSE141051 |
Absolute nucleosome occupancy map for the Saccharomyces cerevisiae genome [ODM-seq] |
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| Relations |
| BioSample |
SAMN13390201 |
| SRA |
SRX7218269 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4193155_WT1_xl_GpC_2U_180min.bedgraph.gz |
3.9 Mb |
(ftp)(http) |
BEDGRAPH |
| GSM4193155_WT1_xl_GpC_2U_180min.tsv.gz |
4.6 Mb |
(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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