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| Status |
Public on Feb 25, 2017 |
| Title |
ChIP-seq RNA PolII DRB [Dnmt3b KO (cl. B126)] |
| Sample type |
SRA |
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| Source name |
Mouse embryonic stem cells
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| Organism |
Mus musculus |
| Characteristics |
strain background: 129/Ola
age: E14
genotype/variation: Dnmt3b ko
cell type: Mouse embryonic stem cells (mESCs)
transfected with: DRB (8h)
treated with: DRB for 8hrs
chip antibody: RNA Polymerase (sc-899)
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| Treatment protocol |
Generation of Dnmt3b KO ESCs was performed using TALEN technology. In brief, cells were transfected with the two TALEN constructs targeting Exon 17 of murine Dnmt3b and after 16 hours were seeded as a single cell. After 1 week, clones were screened by western blot analyses. Positive clones were analyzed by genomic sequencing of the TALEN target.
Transfection of mouse ESCs was performed using Lipofectamine™ 2000 Transfection Reagent in according to manufacturer’s protocol using equal amount of each plasmid (5 g) in multiple transfections. For the Setd2 knockdown, cells were transfected with the specific shRNA constructs, and maintained in medium with puromycin selection (1g/ml) for 48h.
For RNA PolII elongation inhibition, wt and Dnmt3b-/- ESCs were treated with DRB at the concentration of 75mM for 8h.
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| Growth protocol |
Mouse embryonic stem cells E14 were grown on 0.1% gelatin-coated plates and maintained in DMEM (4.5g/L D-Glucose), supplemented with 15% heat-inactivated FBS, 0.1mM NEAA, 1mM Sodium Pyruvate, 0.1mM 2-Mercaptoethanol, 25U/ml penicillin, 25 μg/ml streptomycin and 1,500U/ml LIF
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
For ChIP-seq, approximately 2 x 10E7 cells were cross-linked by addition of formaldehyde to 1% for 10 min at RT, quenched with 0.125 M glycine for 5 min at RT, and then washed twice in cold PBS. The cells were resuspended in Lysis Buffer 1 (50 mM Hepes-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 10% Glycerol, 0.5% NP-40, 0.25% Triton X-100 and protease inhibitor) to disrupt the cell membrane and in Lysis Buffer 2 (10 mM Tris-HCl pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA and protease inhibitor) to isolate nuclei. The isolated nuclei were then resuspended in SDS ChIP Buffer (20 mM Tris-HCl pH 8.0, 10 mM EDTA, 1% SDS and protease inhibitors). Extracts were sonicated using the Bioruptor H Twin (Diagenode) for 2 runs of 10 cycles [30 sec ‘‘ON’’, 30 sec ‘‘OFF’’] at high power setting. Cell lysate was centrifuged at 12,000 g for 10 min at 4°C. The supernatant was diluted with ChIP Dilution Buffer (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 2 mM EDTA, 1% Triton) before immunoprecipitation step. Magnetic beads (Dynabeads® Rat Anti-Mouse IgM for anti-PolII-phospho-S5, Dynabeads®Protein G for all the other ChIPs, Life Technologies) were saturated with PBS/1% BSA and the samples were incubated with 2 micrograms of antibody overnight at 4°C on a rotator. Next day samples were incubated with saturated beads for two hours at 4°C on a rotator. Successively immunoprecipitated complexes were washed five times with RIPA buffer (50 mM Hepes-KOH pH7.6, 500 mM LiCl, 1 mM EDTA, 1% NP-40, 0,7% Na-Deoxycholate) at 4°C for 5 minutes each on a rotator. Elution Buffer was added and incubated at 65°C for 15 minutes. The decrosslinking was performed at 65°C overnight. Decrosslinked DNA was purified using QIAQuick PCR Purification Kit (QIAGEN) according to the manufacture’s instruction.
For ChIP-seq, approximately 10 ng of purified ChIP DNA were end-repaired, dA-tailed, and adapter-ligated using the NEBNext ChIP-Seq Library Prep Master Mix Set (NEB), following manufacturer instructions.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
Basecalls performed using CASAVA version 1.8
ChIP-seq reads were aligned to the mm9 genome assembly using Bowtie v0.12.7 using the following parameters : -q --max /dev/null -v 1 -S --sam-nohead -m 1
Data were filtered using the following specifications: duplicate reads were filtered out
Genome_build: mm9
Supplementary_files_format_and_content: bedGraph files were performed by using MACS tool
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| Submission date |
Sep 09, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Francesco Neri |
| E-mail(s) |
[email protected]
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| Organization name |
University of Torino
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| Street address |
Via Nizza 52
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| City |
Torino |
| State/province |
Italy |
| ZIP/Postal code |
10126 |
| Country |
Italy |
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| Platform ID |
GPL19057 |
| Series (2) |
| GSE72853 |
Intragenic DNA methylation prevents cryptic transcription initiations on gene bodies [ChIP-seq] |
| GSE72856 |
Intragenic DNA methylation prevents cryptic transcription initiations on gene bodies |
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| Relations |
| BioSample |
SAMN04044209 |
| SRA |
SRX1213678 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1873397_PolII_KO_DRB.bedGraph.gz |
778.8 Mb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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