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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Oct 01, 2022 |
| Title |
ES_ChIP_S5P_1 |
| Sample type |
SRA |
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| Source name |
mES-cells
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| Organism |
Mus musculus |
| Characteristics |
protocol: ChIP-seq
assay: S5P (Abcam, ab5131)
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
ES cells were fixed with 1% formaldehyde in DMEM at 37 °C for 10 min, neutralized in DMEM containing 125 mM glycine at room temperature for 5 min and washed with PBS three times. Fixed cells were collected with 2ml soft lysis buffer (50 mM Tris-HCl, pH 8.0, 10 mM EDTA and 0.1% NP-40 10% Glycerol) and centrifuged at 3000 rpm for 15 min. The cell pellet was resuspended in 100µl SDS lysis buffer (1% SDS, 10mM EDTA, 50mM Tris-HCl, pH 8.0), and sonicated. Soluble chromatin was diluted in 0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl, pH 8.0, 167mM NaCl) and incubated with 2µg anti-Pol II Ser5P (Abcam, ab5131), anti-Pol II Ser2P (Abcam, ab5095) antibodies or IgG at 4 °C overnight. Bound chromatin was purified on magnetic beads after washings in low salt buffer (20 mM Tris-HCl, pH 8.0, 2 mM EDTA, 150 mM NaCl, 0.1% SDS, 1% Triton X-100), high salt buffer (20 mM Tris-HCl, pH 8.0 ,2 mM EDTA, 500 mM NaCl, 0.1% SDS, 1% Triton X-100) and LiCl buffer (250 mM LiCl, 1% NP-40, 1% sodium deoxycholate). Chromatin was eluted in 1% SDS, 100mM NaHCO3 at 65 °C, reverse-crosslinked and was purified by phenol-chloroform extraction.
ChIP–seq libraries were prepared using a NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB) and the library quality was assessed using a 2100 Bioanalyzer (Agilent). Samples were paired-end sequenced at PE150 on an Illumina Hiseq 4000.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
The mouse reference genome and annotations were downloaded from Ensembl (version GRCm38.dna.primary_assembly.fa and GRCm38.101.gtf).
Paired-end sequencing reads were trimmed using cutadapt (version 3.4) with parameters -a CTGTCTCTTATA -A CTGTCTCTTATA --pair-filter=any --minimum-length=20.
Trimmed reads were mapped to the reference genome using bowtie2 (version 2.3.5) with parameters --end-to-end --very-sensitive --no-unal --no-mixed --no-discordant -I 10 -X 500.
Aligned reads were filtered by quality using samtools (version 1.3) with parameter -q 12. Read pairs were read in to R (version 3.6.3) using readGAlignmentPairs from the GenomicAlignment package (version 1.22.0) and fragments were filtered for fragments that have both unique start and end positions.
Coverage vectors were created using the coverage function from GenomicRanges package (version 1.38.0) and were normalized by the number of fragments and multiplied by a million. Normalized coverage were exported as bigwig files using the rtracklayer package (version 1.46.0).
Genome_build: GRCm38
Supplementary_files_format_and_content: bigwig files of normalized coverage
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| Submission date |
Jan 31, 2022 |
| Last update date |
Oct 02, 2022 |
| Contact name |
Tamas Schauer |
| E-mail(s) |
tamas.schauer@helmholtz-munich.de
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| Organization name |
Helmholtz Zentrum München
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| Department |
Institute of Epigenetics and Stem Cells
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| Street address |
Feodor-Lynen-Straße 21
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| City |
Munich |
| ZIP/Postal code |
81377 |
| Country |
Germany |
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| Platform ID |
GPL24247 |
| Series (2) |
| GSE195830 |
Characterization and function of transcriptional elongation during zygotic genome activation in mouse embryos [ChIP-seq] |
| GSE195840 |
Characterization and function of transcriptional elongation during zygotic genome activation in mouse embryos |
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| Relations |
| BioSample |
SAMN25520975 |
| SRA |
SRX13999606 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5851556_ES_ChIP_S5P_1_norm_coverage.bw |
228.9 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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