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| Status |
Public on Aug 17, 2012 |
| Title |
EAR003_siRNA-tdg |
| Sample type |
SRA |
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| Source name |
Embryonic stem cells, TDG siRNA, 5fC
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| Organism |
Mus musculus |
| Characteristics |
cell line: J1
strain/background: 129S4/SvJae
cell type: embryonic stem cells
sirna: TDG-KD
pulldown target: 5-formylcytosine (5fC)
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| Treatment protocol |
RNA interference experiments were performed in J1 ES cells without feeders with three rounds of transfections with siRNA every second day. In the first day, 1 x 10^5 cells were seeded per well (3.8cm2) of a 12-well plate and adherent cells were transfected the next day with 20 pmol siRNA: 3 mL Lipofectamine™2000 complexes in media without antibiotics according to the manufacturer's instructions. After 8 hours, fresh media with antibiotics was added to the cells. This procedure was repeated 48 and 96 hours after the first transfection, both these times on cells in suspension. Cells were passaged and transfections were scaled up when necessary. Transfections were done with Dharmacon (Thermo Fisher Scientific) siGENOME SMARTpool against mouse Tdg (catalogue no. M-040666-01; gaagugcaguauacauuug, gaguaaagguuaagaacuu, caaagaagauggcuguuaa, gcaaggaucugucuaguaa) and siGENOME non-targeting siRNA#2 (catalogue no. D-001210-02; sequence not available). Cells were harvested after three rounds of transfection for DNA/RNA isolation.
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| Growth protocol |
Cell culturing was done on the J1 ES cell line (129S4/SvJae), purchased from ATCC (catalogue no. SCRC-1010) and grown on a γ-irradiated pMEF feeder layer at 37°C and 5% CO2 in complete ES medium (DMEM 4,500 mgl-1 glucose, 4 mM L-glutamine and 110 mg l-1 sodium pyruvate, 15% fetal bovine serum, 100 U of penicillin/100 mg of streptomycin in 100 ml medium, 0.1mM non-essential amino acids, 50mM β-mercaptoethanol, 103U LIF ESGRO).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
DNA was extracted from cells using the AllPrep DNA/RNA Kit from Qiagen (UK) following the manufacturer's instructions.
We devised a method to map 5-formylcytosine (5fC) by linking a biotin tag to 5fC for pulldown and high-throughput sequencing.
After chemical pulldown, the ends of the DNA fragments were repaired and paired-end sequencing-specific adaptors (Illumina) were ligated using the NEBNext DNA Sample Prep Reagent Set 1 (NEB). Following adaptor ligation, DNA and 2 μg poly-dI:dC were incubated with 50 μg streptavidin-coated magnetic beads (MagneSphere Promega) in 50 μL 2x binding buffer (10 mM Tris pH 7.5, 1 mM EDTA, 2M NaCl, 0.1% TWEEN) for 15 min at room temperature. Beads were washed with 5x 500 μL binding buffer and transferred into a new eppendorf. For elution, beads were incubated with 100 uL elution solution (95% formamide, 10 mM EDTA and 400 nM biotin) at 90°C for 5 min and the eluant was collected and immediately placed on ice. This step was repeated to elute any residual DNA. Eluted DNA was then precipitated in ethanol and the DNA pellet was resuspended in 15 uL ddH2O. Fragments were amplified with 16 cycles using adaptor-specific primers (Illumina); fragments ranging between 200 and 500 bp in size were gel-purified before cluster generation and sequencing. Sequencing was done on an Illumina Genome Analyzer GAIIX using Cluster Generation v4 and 5 chemistries as well as Sequencing by Synthesis Kit v5. Data collection was performed using Sequencing Control Software v2.6 and 2.9. Real-time Analysis (RTA) 1.6 and 1.9 were used for base calling.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina Genome Analyzer IIx |
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| Data processing |
Basecalls were performed using CASAVA pipeline version 1.5-1.7.
Reads were aligned to the mouse reference genome (UCSC version mm9) using bwa 0.6.1 with default settings.
Reads mapped with MAPQ < 15 were filtered out.
Genomic regions enriched in 5fC were identified with the program rseg (Song and Smith, Bioinformatics 2011) in mode 2 using the input library as control and setting the bin size to 100 bp.
Genome_build: mm9
Supplementary_files_format_and_content: Domain files in bed format produced by rseg (Song and Smith, Bioinformatics 2011). Each file contains the coordinates of the 5fC-enriched regions (where 4th column= 'ENRICHED') and background regions (where 4th column= 'BACKGROUND'). Please refer to the rseg manual for further details (http://smithlab.usc.edu/histone/rseg/).
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| Submission date |
Aug 15, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Dario Beraldi |
| E-mail(s) |
[email protected]
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| Organization name |
Cambridge Research Institute
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| Street address |
Robinson Way
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| City |
Cambridge |
| ZIP/Postal code |
CB2 0RE |
| Country |
United Kingdom |
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| Platform ID |
GPL11002 |
| Series (1) |
| GSE40148 |
Genome-wide distribution of 5-formylcytosine in ES cells is associated with transcription and depends on TDG |
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| Relations |
| SRA |
SRX177652 |
| BioSample |
SAMN01120142 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM986293_ear003_j1_sirna_tdg.domains.bed.gz |
1.2 Mb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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