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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Feb 25, 2017 |
| Title |
RNAseq_ESC_KO_DRB_3h |
| Sample type |
SRA |
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| Source name |
E14 mESC Dnmt3b ko, DRB, 3h
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| Organism |
Mus musculus |
| Characteristics |
cell line: E14
cell type: mouse embryonic stem cells
strain/background: 129/Ola
genotype/variation: Dnmt3b ko
cell treatment: DRB
time point: 3h
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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 according to manufacturer's protocol using equal amount of each plasmid (5 ug) in multiple transfections. For the Setd2 knockdown, cells were transfected with the specific shRNA constructs, and maintained in medium with puromycin selection (1ug/ml) for 48h.
For half-life measurements, wt and Dnmt3b-/- ESCs were treated with DRB at the concentration of 75uM for the indicated time.
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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 ug/ml streptomycin and 1,500U/ml LIF.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted by using TRIzol reagent (Invitrogen).
For Ribo- RNA-seq library preparation, 2.5 ug of total RNA were depleted of ribosomal RNA using the RiboMinus Eukaryote System v2 kit (Invitrogen), following manufacturer instructions. Ribo- RNA was resuspended in 17ul of EFP buffer (Illumina), heated to 94°C x 8', and used as input for First Strand synthesis, using the TruSeq RNA Sample Prep kit, following manufacturer instructions. PolyA+ RNA-seq were performed by using the TruSeq RNA Sample Prep kit, following manufacturer instructions.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiScanSQ |
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| Description |
PolyA enriched
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| Data processing |
Basecalls performed using CASAVA version 1.8.
For RNA-seq, reads were mapped on mm9 using TOPHAT v2.0.6 with the following parameters: --min-anchor=5 --min-isoform-fraction=0.01 --max-multihits=10 --bowtie1
Genome_build: mm9 (MGSCv37)
Supplementary_files_format_and_content: bedGraph files were performed by using Wiggles tool.
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| Submission date |
Dec 09, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Francesco Neri |
| E-mail(s) |
francesco.neri@unito.it
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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 |
GPL16173 |
| Series (2) |
| GSE72855 |
Intragenic DNA methylation prevents cryptic transcription initiations on gene bodies [RNA-seq] |
| GSE72856 |
Intragenic DNA methylation prevents cryptic transcription initiations on gene bodies |
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| Relations |
| BioSample |
SAMN06128404 |
| SRA |
SRX2410343 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2422508_RNAseq_ESC_KO_DRB_3h.bedGraph.gz |
75.4 Mb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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