|
| Status |
Public on Mar 07, 2016 |
| Title |
SRSF5_iCLIP_mm9_Rep2 |
| Sample type |
SRA |
| |
|
| Source name |
P19
|
| Organism |
Mus musculus |
| Characteristics |
cell line: P19 embryonal carcinoma cell line
fusion protein expression: SRSF5-GFP
antibody: anti-EGFP (D. Drechsel, MPI-CBG)
barcode sequence: TGGC
|
| Treatment protocol |
Cells were washed with PBS and irradiated with 254 nm UV light to crosslink proteins to nucleic acids.
|
| Growth protocol |
Cells were grown in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal calf serum and pennicilin/streptomycin antibiotics at 37˚C with 5% CO2.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were lysed and treated with RNase I. GFP-tagged proteins were immunoprecipitated with Gamma binding beads coupled to goat anti-EGFP antibody (D. Drechsel, MPI-CBG).
After stringent washing, RNA was dephosphorylated and ligated to a 3' linker. Protein-RNA complexes were resolved by denaturing gel electrophoresis and transfered to nitrocellulose membrane. A membrane region above the expected size of the protein was cut and RNA was released by Proteinase K treatment. RNA was reverse transcribed using reverse primer containing a barcode sequence. cDNA was separated into 3 size fractions by denaturing gel electrophoresis and circularized by ssDNA circligase. A specific oligonukelotide was annealed to create a restriction site for BamHI and cDNA was linearized by digest with BamHI. Linearized cDNA was PCR amplifiied with Solexa sequencing primers to create the final libraries. For more details see: König et al. (2010): iCLIP reveals the function of hnRNP particles in splicing at individual nucleotide resolution. Nat Struct Mol Biol PMID:20601959
|
| |
|
| Library strategy |
RIP-Seq |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
processed data file: mSRSF5_iCLIP_all_mm9_iCLIP_tags.bed
processed data file: mSRSF5_iCLIP_all_mm9_iCLIP_tag_clusters.bed
|
| Data processing |
Basecalls performed using CASAVA version 1.4
For detail on data processing see: König et al. (2010): iCLIP reveals the function of hnRNP particles in splicing at individual nucleotide resolution. Nat Struct Mol Biol PMID:20601959
Genome_build: mm9
Supplementary_files_format_and_content: .bed files containing the genomic coordinates for significant CLIP tags for individual replicates or combined replicates (3-6 replicates).
Supplementary_files_format_and_content: .bed files containing the genomic coordinates for significant CLIP tag clusters from combined replicates.
|
| |
|
| Submission date |
Jun 09, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Michaela Müller-McNicoll |
| E-mail(s) |
[email protected]
|
| Phone |
+49 69 798 42079
|
| Organization name |
Goethe University Frankfurt
|
| Department |
Institute for Cell Biology and Neuroscience
|
| Lab |
RNA Regulation Group
|
| Street address |
Max-von-Laue-Str. 13
|
| City |
Frankfurt am Main |
| ZIP/Postal code |
60438 |
| Country |
Germany |
| |
|
| Platform ID |
GPL13112 |
| Series (2) |
| GSE69689 |
Transcriptome-wide mapping of RNA:protein interactions of seven SR proteins and NXF1 in P19 cells by iCLIP |
| GSE69734 |
SR protein family in P19 cells |
|
| Relations |
| BioSample |
SAMN03766054 |
| SRA |
SRX1054352 |
