|
| Status |
Public on May 01, 2015 |
| Title |
plus-plus-2 |
| Sample type |
SRA |
| |
|
| Source name |
E13.5-MEFs
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57bl/6
cell type: MEF
developmental stage: E13.5
usp28-genotype: +/+
overexpression product: none
ras-transformed: no
replicate: 2
|
| Treatment protocol |
Cells were immortalized with pRetroSuper-shp19ARF. Partly, cells were transformed with pBabe-HRasG12V and infected with pWZL-USP28 or pWZL-GFP.
|
| Growth protocol |
MEFs were grown in DMEM (Sigma) supplemented with 10% FBS (Biochrom AG) and 1% penicillin/streptomycin (Sigma).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using the RNeasy Mini Kit (Qiagen) including on-column DNase digestion. Poly-A RNA was isolated from total RNA using the NEBNext Poly(A)mRNA Magnetic Isolation Module (E7490).
Libraries for the RNA-seq samples were constructed using the NEBNext Library Prep Kit (6100) or NEBNext Ultra RNA Library Prep Kit for Illumina (E7530) following the instruction manual. Briefly, poly-A RNA was fragmented to generate 200 nucleotides long fragments. First and second strand cDNA synthesis was performed and the resulting cDNA was end-repaired, ligated to NEBNext adaptors. The cDNA was size selected using agarose gels or Agencourt AMPure XP Beads (Beckman Coulter) and amplified by PCR. For the resulting RNA-seq libraries, amplicon sizes and quantities were determined using the Biorad Experion system. Libraries were sequenced on an Illumina Genome Analyzer IIx following the manufacture’s instructions.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina Genome Analyzer IIx |
| |
|
| Data processing |
Basecalling was performed with the real time analysis (RTA) package within the Genome Analyzer Sequencing Control Software (SCS2.10).
Demultiplexing and generation of Fastq files was done with the CASAVA software only considering high quality sequences (PF-cluster).
Overall sequencing quality was checked with the FastQC script.
Reads were aligned to the murine genome (mm9) with BOWTIE v0.12.8.
For RNAseqs, differential expression and statistical inference was done using EdgeR.
Genome_build: mm9
Supplementary_files_format_and_content: For RNAseq, the txt-files contain tables showing the differential expression (as log FoldChange, in column) of ensemble annotated genes (ensemble gene ids, in rows) between samples (=experimental conditions) and the calculated p-value and/or adjusted p-value for each FoldChange.
|
| |
|
| Submission date |
Jul 11, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Martin Eilers |
| Organization name |
University of Wuerzburg
|
| Department |
Chair for Biochemistry and Molecular Biology
|
| Lab |
Martin Eilers
|
| Street address |
Am Hubland
|
| City |
Wuerzburg |
| ZIP/Postal code |
97074 |
| Country |
Germany |
| |
|
| Platform ID |
GPL11002 |
| Series (1) |
| GSE59354 |
Dual regulation of Fbw7 function and oncogenic transformation by Usp28 |
|
| Relations |
| BioSample |
SAMN02910232 |
| SRA |
SRX651529 |
