|
| Status |
Public on Sep 13, 2013 |
| Title |
MEF_ko_rep1 |
| Sample type |
SRA |
| |
|
| Source name |
Mouse Embryonic Fibroblasts
|
| Organism |
Mus musculus |
| Characteristics |
cell type: MEF
treatment: Prmt5 depletion
developmental stage: E14.5
genotype: Prmt5 F/FER
treatment: 4-OHT
strain: mixed C57BL/6 X 129S1/SvlmJ
|
| Treatment protocol |
PRMT5F/F ER day 4 NPCs and MEFs were treated with either 50nM 4-OHT or the equivalent volume of ethanol for 24 hours before splitting to induce PRMT5 knockout.
|
| Growth protocol |
Neurosphere cultures were established as previously described. Briefly, E14.5 embryos were harvested and cortices carefully dissected in ice-cold PBS and incubated in trypsinfor 10 min at 37°C. The tissue was then mechanically dissociated into single cell suspension and passed through a 40 μm cell strainer into complete NSC medium, 1% penicillin-streptomycin, 20 ng/ml recombinant human epidermal growth factor and 20 ng/ml recombinant human fibroblast growth factor-basic. Mouse Embryonic Fibroblasts (MEFs). Primary MEFs were prepared from E14.5 embryos as previously described 48 and maintained in a humidified 5% CO2 atmosphere at 37°C in DMEM supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. To induce PRMT5 knockout, MEFs (passage 1) were grown to confluence in 15 cm-dishes.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was harvested using Trizol reagent.
Illumina TruSeq RNA Sample Prep Kit v2(Cat#FC-122-1001) was used with 1 ug of total RNA for the construction of sequencing libraries.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Data processing |
Illumina Casava v1.8 used for basecalling.
Sequences were mapped to mm9 using TopHat v2.03 and Bowtie 2.0.0.6 with parameters -m 1 -n 2 --no-coverage-search --library-type fr-unstranded --solexa-quals
FPKM values were computed using cuffdiff 2.02 with parameters -u --total-hits-norm -N
To determine differential splicing events, MATS 3.0.6 beta was used counting junction reads and reads falling into the tested region within ENSEMBL v65 gene definitions. Matching embryos were analysed individually and only significant events occurring in at least two replicates were considered. Splicing events were labelled significant if the sum of the reads supporting a specific event exceeded 10 reads, the p-value was lower than 0.05 and the minimum inclusion level difference as determined by MATS was higher than 0.2. All other parameters were left at the default value.
Genome_build: mm9
Supplementary_files_format_and_content: tab-delimited text files include FPKM values and differential splicing results for each sample
|
| |
|
| Submission date |
Mar 19, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Julius Müller |
| Organization name |
A*Star Singapore
|
| Department |
IMCB
|
| Lab |
Ernesto Guccione
|
| Street address |
61 Biopolis Drive
|
| City |
Singapore |
| ZIP/Postal code |
138673 |
| Country |
Singapore |
| |
|
| Platform ID |
GPL13112 |
| Series (2) |
| GSE45284 |
Regulation of constitutive and alternative splicing by PRMT5 reveals a role for Mdm4 pre-mRNA in sensing defects in the spliceosomal machinery (RNA-Seq) |
| GSE45285 |
Regulation of constitutive and alternative splicing by PRMT5 reveals a role for Mdm4 pre-mRNA in sensing defects in the spliceosomal machinery |
|
| Relations |
| SRA |
SRX252197 |
| BioSample |
SAMN01984508 |
