|
| Status |
Public on Feb 25, 2017 |
| Title |
DECAPseq_ESC_WT_shSetd2 |
| Sample type |
SRA |
| |
|
| Source name |
E14 mESC wt, Setd2 shRNA transfection
|
| Organism |
Mus musculus |
| Characteristics |
cell line: E14
cell type: mouse embryonic stem cells
strain/background: 129/Ola
genotype/variation: wt
cell treatment: transfection (Setd2 shRNA)
|
| 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.
|
| 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.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted by using TRIzol reagent (Invitrogen).
Total RNA (or polyA RNA enriched using The NEBNext Poly(A) mRNA Magnetic Isolation Module kit (NEB) following manufacturer instructions or cytosolic RNA) was first ribo- and URNA-depleted and then was chemically fragmented by using First Strand buffer of the SuperScript® II Reverse Transcriptase (Invitrogen). The fragmented RNA was Dephosphorylated of natural 5' and fragmentation-derived 3' phosphate by using Antarctic Phosphatase (AP, NEB). Dephosphorylated RNA was then treated with RNA 5' Pyrophosphohydrolase (RppH, NEB) in 1X Thermopol buffer (NEB) (for decapping and pyrophosphate removal from the 5' end of RNA to leave a 5' monophosphate RNA). 5' adapter was ligated, and 3' adapter was added by reverse transcription.
|
| |
|
| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
Ribo and URNA depleted
|
| Data processing |
Library strategy: Decapping-sequencing (DECAP-seq)
Basecalls performed using CASAVA version 1.8.
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.
|
| |
|
| Submission date |
Dec 09, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Francesco Neri |
| E-mail(s) |
francesco.neri@unito.it
|
| Organization name |
University of Torino
|
| Street address |
Via Nizza 52
|
| City |
Torino |
| State/province |
Italy |
| ZIP/Postal code |
10126 |
| Country |
Italy |
| |
|
| Platform ID |
GPL19057 |
| Series (2) |
| GSE72854 |
Intragenic DNA methylation prevents cryptic transcription initiations on gene bodies [DECAP-seq] |
| GSE72856 |
Intragenic DNA methylation prevents cryptic transcription initiations on gene bodies |
|
| Relations |
| BioSample |
SAMN06129861 |
| SRA |
SRX2410620 |
