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GEO help: Mouse over screen elements for information. |
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| Status |
Public on May 15, 2019 |
| Title |
2.7kb mixed_population nodox 10nM repl1 |
| Sample type |
SRA |
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| Source name |
2.7kb mixed_population nodox 10nM
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| Organism |
Mus musculus |
| Characteristics |
cell type: Embyonic stem cell
clone: 2.7kb teto integrations
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| Treatment protocol |
To induce nuclear translocation of the rTetR-Dam fusion protein to the nuclei, mESC were trypsinized and directly seeded in culture medium containing 4-hydroxy-tamoxifen (4-OHT) at the concentrations indicated for 18 hours. Binding of the Dam fusion protein to the TetO arrays was induced by simultaneously adding 2.5 μg/ml doxycycline (Dox).
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| Growth protocol |
Cells were cultured on gelatin-coated culture plates in Dulbecco Modified Eagle’s medium (Sigma) in the presence of 15% foetal calf serum (Eurobio Abcys), 100 µM β-mercaptoethanol, 20 U/ml leukemia inhibitory factor (Miltenyi Biotec, premium grade) in 8% CO2 at 37°C. After insertion of the rTetR-Dam vector (see below), cells were cultured in the presence of 250 µg/mL hygromycin.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
3x106 cells were harvested using trypsin after 18 hours of induction with tamoxifen +/- doxycyclin. Genomic DNA was extracted using the Qiagen blood and tissue kit adding RNaseA in step 1. Genomic DNA was eluted in 80ul ddH2O. DNA concentration was measured using the Qbit DNA Broad Range kit
Genomic DNA (100ng input) was treated with Shrimp Alkaline Phosphatase treatment (NEB, 1U), followed by DpnI digestion (ThermoFisher Scientific, 10U), A-tailing (0.6mM final dATP, 5U Klenow exo-, ThermoFisher Scientific), and UMI adapters ligation (30U T4 DNA ligase, PEG4000, ThermoFisher Scientific) performed within the same tube and buffer (Tango 1X, ThermoFisher Scientific) by heat inactivating each enzymatic step followed by adjustment with the reagents required for the next step, Ligation reactions were treated with Exonuclease I (20U, ThermoFisher Scientific ) then purified using AMPureXP beads (1:0.8 ratio, Agencourt) and the second sequencing adapter was tagged using heat denaturation and second strand synthesis (5U T4 DNA Polymerase, ThermoFisher Scientific). The tagging reaction was purified using AMPure XP beads (1:1 ratio) followed by a final library amplification (10 cycles) using 1U of Phusion polymerase, 2μl 10 μM DAM_UMIindex_PCR was tagged using heat denaturation and second strand synthesis (5U T4 DNA Polymerase, ThermoFisher Scientific). The tagging reaction was purified using AMPure XP beads (1:1 ratio) followed by a final library amplification (10 cycles) using 1U of Phusion polymerase, 2μl 10 μM DAM_UMIindex_PCR.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
processed data file:
Exp1374X9_1374X10_Enrichment_dedupON_singleFragment_20steps_before_ration.txt
barcoded.1374F10
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| Data processing |
library strategy: damC library preparation
Single-end 76bp damC-seq reads from each sample were mapped to the mouse genomes using the QuasR software (v 1.20.0, qAlign function, default settings).
Deduplication based on UMIs was done using a custom script on bam files (UMIs are contained in the readname of each read in each fastq file)
damC signal quantification was done using qAlign (from QuasR, v 1.20.0) using as query a GRanges object containing the position of GATC sites shifted by 5bps upstream (plus strand) and downstream (minus strand). Library sizes are rescaled to have 10M reads mapped to the query and a pseudocount of 0.2 is added
Running average of the signal was done prior enrichment calculation using a custom script and using 21 steps
Enrichment was then calculated as described in the paper
Genome_build: mm9
Supplementary_files_format_and_content: tab-delimited text files including genomic coordinates and enrichments
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| Submission date |
Mar 07, 2019 |
| Last update date |
Jun 01, 2024 |
| Contact name |
Luca Giorgetti |
| Organization name |
Friedrich Miescher Institute for Biomedical Research
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| Street address |
Fabrikstrasse 24
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| City |
Basel |
| ZIP/Postal code |
4056 |
| Country |
Switzerland |
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| Platform ID |
GPL19057 |
| Series (2) |
| GSE128013 |
DamC reveals principles of chromatin folding in vivo without crosslinking and ligation [damC] |
| GSE128017 |
DamC reveals principles of chromatin folding in vivo without crosslinking and ligation |
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| Relations |
| BioSample |
SAMN11083019 |
| SRA |
SRX5493655 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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