|
| Status |
Public on Mar 07, 2016 |
| Title |
KD SRSF4 cyto Replicate A |
| Sample type |
SRA |
| |
|
| Source name |
P19
|
| Organism |
Mus musculus |
| Characteristics |
cell line: P19 embryonal carcinoma cell line
genotype/variation: KD SRSF4
cell fraction: cytoplasm
|
| Treatment protocol |
Seven different SR proteins were knocked down using costum-made esiRNAs and Lipofectamin2000. An esiRNA against GFP was used for mock transfections. Transfections were performed for 36 hours to minimize secondary effects on gene expression.
|
| 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 |
polyA RNA |
| Extraction protocol |
P19 Control and Knockdown cells were harvested and separated into whole cell lysates and cytoplasmic fractions. RNA was isolated using TRIzol. PolyA+ RNA was enriched with MicroPoly(A) Purist Kit (Ambion.
mRNA isolation from 1ug total RNA was done by poly-dT enrichment using the NEBNext Poly(A) mRNA Magnetic Isolation Module according to the manufacturers instructions. Library was prepared using TruSeq library prep kit (Illlumina).
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
PolyA+ RNAseq cyto
SRSF4VsGFP_CytoVsWholecell_CuffDiff.txt
Cyto_Gene_FPKMs.txt
|
| Data processing |
Basecalls performed using CASAVA version 1.8.2
Remaining adaptor sequences were trimmed from reads using cutadapt -m 20 -q 25; For MISO all reads were trimmed to 75-bp length.
For Cufflinks and DEX-Seq RNA-seq reads were aligned to mm10 using Tophat v2.0.9 coupled with Bowtie v2.1.0. For MISO reads were mapped to mm9 using TopHat (v2.0.10) with the option -g1.
FPKMs were calculated using Cufflinks v2.1.1; Splicing changes were calculated with DEX-Seq and MISO.
Genome_build: Cufflinks: mm10; Ensembl v73. gtf-used for expression profiling was retrieved with ensembl-ids from uscc table browser. DEX-Seq: mm10; ENSEMBL v66. MISO: mm9
Supplementary_files_format_and_content: Cufflinks: Tab-delimited text files containing gene counts of all knockdown experiments separate in cytoplasmic and whole cell samples; FPKM values of all knockdown experiments separate in cytoplasmic and whole cell samples; FPKM values of separate knockdown experiments in cytoplasmic and whole cell samples. DEX-Seq: Text files containing read counts in exon counting bins comparing KD of SR proteins to control. MISO: Text files containing changes in expression in annotated alternative last exons (ALE) and TandemUTRs including Percent Spliced In (PSI) values and confidence intervals calculated and compared between KD samples and controls.
|
| |
|
| Submission date |
Jun 10, 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) |
| GSE69733 |
Knockdown of SR protein family in P19 cells and RNA-seq of cytoplasmic and whole cell polyA+ RNA |
| GSE69734 |
SR protein family in P19 cells |
|
| Relations |
| BioSample |
SAMN03768399 |
| SRA |
SRX1055030 |
