|
| Status |
Public on Apr 21, 2011 |
| Title |
mES_RNA_Seq_shRNA3387 |
| Sample type |
SRA |
| |
|
| Source name |
ES cells, Tet1 shRNA3387
|
| Organism |
Mus musculus |
| Characteristics |
cell line: E14
cell type: mouse embryonic stem (MES) cells
shrna treatment: Tet1 shRNA3387
|
| Treatment protocol |
E14 ES cells were infected with Tet1 shRNA3387 containing lentivirus and harvested after 4-days selection with puromycin. Total RNA was extracted using TRIzol reagent (Invitrogen) and genomic DNA was removed by DNaseI digestion. The mRNA was further purified using Dynabeads mRNA purification kit (Invitrogen) and used to synthesize the first strand cDNA using ImPro-IITM Reverse Transcription System (Promega). The second strand cDNA was generated using DNA Pol I (Invitrogen) and the final double-stranded DNA (dsDNA) was purified using QIAquick PCR purification kit (Qiagen) and quantified by Qubit fluorometer (Invitrogen). 300 ng of dsDNA of each sample was fragmented by sonication and used to generate the sequencing library.
|
| Growth protocol |
E14 ES cells were feeder-free cultured.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
DNA was end repaired using the End-It DNA End-repair kit (Epicentre), and purified with the QiaQuick PCR purification kit. DNA was treated with Klenow exo- (NEB) in NEB buffer 2 with 1mM ATP. After incubating at 37oC for 50min, the "A" base added DNA was purified with the QiaQuick PCR purification kit. Illumina adapters were ligated using quick T4 DNA ligase. The final DNA was purified with the QiaQuick PCR purification kit and amplified using Illumina sequencing primers for 16-18 cycles.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina Genome Analyzer |
| |
|
| Description |
mRNA-seq
cDNA reverse transcribed from the total mRNA of Tet1 shRNA3387-treated cells.
|
| Data processing |
We used Off-Line Basecaller software (Illumina v1.8) to analyze images and call bases, and used the CASAVA package (Illumina v1.6) to generate read sequences and align to mm9. Since the DNA fragment size in our library is mainly from 200-300bp, we then extended the sequence to 250bp downstream of the 5' end of reads and converted the sorted file into bed format.
|
| |
|
| Submission date |
Apr 11, 2011 |
| Last update date |
May 15, 2019 |
| Contact name |
Feizhen Wu |
| E-mail(s) |
wufz@fudan.edu.cn
|
| Phone |
86-21-54237821
|
| Organization name |
Fudan Univ, Shanghai, China
|
| Department |
Institutes of Biomedical Sciences
|
| Lab |
Epigenetics lab
|
| Street address |
Dongan Road 131, Rm 511 Mingdao Building
|
| City |
Shanghai |
| State/province |
Shanghai |
| ZIP/Postal code |
200032 |
| Country |
China |
| |
|
| Platform ID |
GPL9185 |
| Series (2) |
| GSE28500 |
Genome-wide Regulation of 5hmC, 5mC and Gene Expression by Tet1 Hydroxylase in Mouse Embryonic Stem Cells |
| GSE28532 |
Genome-wide Regulation of 5hmC, 5mC and Gene Expression by Tet1 Hydroxylase in Mouse Embryonic Stem Cells (ChIP-seq data) |
|
| Relations |
| SRA |
SRX057752 |
| BioSample |
SAMN00259674 |
| Named Annotation |
GSM706682_mES_RNA_Seq_shRNA3387.bed.gz |
