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| Status |
Public on Nov 27, 2019 |
| Title |
WT5_xl_5_CpG_2U_180min_refill_at_90min_NP-seq |
| Sample type |
SRA |
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| Source name |
WT5_xl_5_CpG_2U_180min_refill_at_90min
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| Organism |
Saccharomyces cerevisiae |
| Characteristics |
methyltransferase: CpG
replicate: WT5
crosslinked: 5 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). 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.
Oxford Nanopore library construction and sequencing For Nanopore sequencing, 1 µg of purified DNA was subjected to 1D native barcoding (Oxford Nanopore, SQK-LSK109). To get up to 1 Gbases coverage per sample, up to five samples were loaded to a MinION flowcell (R9.4.1).
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
MinION |
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| Data processing |
basecalling with Guppy v3.2.2
Library strategy: ODM-seq
ODM-seq analysis for Nanopore-seq data We called the bases with Albacore (Oxford Nanopore Technologies, version 2.3.3) or Guppy (Oxford Nanopore Technologies, version 3.2.2), for WT4 and WT5, respectively, and mapped the reads with minimap2 (version 2.14-r892-dirty, (Li 2018)). For methylation calling we used Nanopolish (version 0.11.0, (Simpson et al. 2017)). At each CpG methylation site, the occupancy is calculated as ratio of unmethylated reads over the sum of methylated and unmethylated reads, ignoring reads where the site has been called "ambiguous" by Nanopolish. We ignored methylation sites with a coverage less than 20. Note that, currently, Nanopolish groups CpG sites within 10 bp into one site, thus having a lower resolution than BS-seq.
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@mpinat.mpg.de
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| Phone |
+49 551 2012809
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| Organization name |
Max-Planck Institute for Multidisciplinary Sciences
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| Department |
Molecular Biology
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| Lab |
Cramer
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| Street address |
Am Faßberg 11
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| City |
Göttingen |
| ZIP/Postal code |
37077 |
| Country |
Germany |
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| Platform ID |
GPL25739 |
| Series (2) |
| GSE132225 |
Absolute nucleosome occupancy map for the Saccharomyces cerevisiae genome |
| GSE141049 |
Absolute nucleosome occupancy map for the Saccharomyces cerevisiae genome [ODM-nanopore-seq] |
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| Relations |
| BioSample |
SAMN13390164 |
| SRA |
SRX7218256 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4193096_WT5_xl_5_CpG_2U_180min_refill_at_90min_NP-seq.bedgraph.gz |
1.9 Mb |
(ftp)(http) |
BEDGRAPH |
| GSM4193096_WT5_xl_5_CpG_2U_180min_refill_at_90min_NP-seq.tsv.gz |
3.0 Mb |
(ftp)(http) |
TSV |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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