|
| Status |
Public on Aug 11, 2014 |
| Title |
3hH89 replicate1 |
| Sample type |
SRA |
| |
|
| Source name |
Serum-starved (72h) Swiss 3T3 fibroblasts, 3h15min H89 treated (10 uM)
|
| Organism |
Mus musculus |
| Characteristics |
cell line: Swiss 3T3 fibroblasts
cell type: fibroblasts
treatment: 72h starvation in the medium containing 0.2% FCS followed by 3h15min treatment with H89 (10 uM)
|
| Treatment protocol |
Serum-straved (72 h) mouse 3T3 fibroblasts we treated with 188.5 nM of anisomycin (Sigma) for 3 or 6 h, with or without 15-min pre-treatment with 10 uM of H89 inhibitor (Santa Cruz Biotechnology).
|
| Growth protocol |
Swiss 3T3 fibroblasts were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% (vol/vol) fetal calf serum (FCS) in the presence of Penicillin and Streptomycin. The cells were serum-deprived for 72 h using DMEM containing 0.2% FCS (vol/vol).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated using Trizol (Invitrogen) according to manufacturer'instructures.
10 μg of total RNA was subjected to two rounds of polyA selection with Dynabeads® mRNA Purification kit (Invitrogen). mRNA was subsequently fragmented by hydrolysis (40 mM TrisOAc pH 8.2, 100 mM KOAc, 150 mM MgOAc) at 94oC for 3 min. 1st strand cDNA synthesis was performed using Superscript III Reverse Transcriptase kit (Invitrogen) with random hexamers priming (Applied Biosystems) in the presence of Actinomycin D (5 ng/ml). Second strand cDNA was synthesized using DNA Pol I, DNA ligase (both Invitrogen) and RNase H (NEB) with random hexamers (Applied Biosystems) in the presence of dUTP. The libraries were prepared by CSF NGS unit (csf.ac.at) using NEBNext® Library Prep Reagent Set for Illumina (NEB).The following TrueSeq barcodes were used: sample 0h_1 TAGCTT, sample 0h_2 ATCACG, sample 1hA_1 GGCTAC, sample 1hA_2 CGATGT.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
Gene expression after 72h starvation in the medium containing 0.2% FCS followed by 3h15min treatment with H89 (10 uM) (replicate1)
|
| Data processing |
Base callling was performed using RTA version 1.17.21.3. Demultiplexing was performed using BamIndexDecoder version V1.13-3-g8bb9b0 from the Illumina2Bam utils.
Following demultiplexing, the reds were trimmed for the barcode sequence and mapped to the mouse genome (mm9) using TopHat (version 1.4.1). Only one mismatch was allowed per 18 bp segment. Only uniquely mapped reads were retained.
We used htseq-count script (using the "union" model) to calculate the number of reads per each of 21608 mouse RefSeq genes. In cases were multiple RefSeq entries corresponded to one gene symbol, RefSeq accessions associated with the longest transcript were retained. Read numbers from htseq-count script were divided by the sum of the exon lengths of each transcript and normalized by the library size.
Genome_build: mm9
Supplementary_files_format_and_content: Tab deliminated files containing RPKM values for mouse RefSeq genes. Each file contains a header with "RefSeq"and "RPKM".
|
| |
|
| Submission date |
Jun 23, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Anna Sawicka |
| E-mail(s) |
anna.sawicka@mpibpc.mpg.de
|
| Organization name |
Max Planck Institure for Biophysical Chemistry
|
| Department |
Dep. of Molecular Biology
|
| Lab |
Cramer
|
| Street address |
Am Fassberg 11
|
| City |
Goettingen |
| ZIP/Postal code |
37077 |
| Country |
Germany |
| |
|
| Platform ID |
GPL13112 |
| Series (2) |
| GSE55784 |
Genome-wide analysis of stress response in mouse fibroblasts |
| GSE58746 |
Transcriptional response to stress in serum deprived mouse fibroblasts in the presence of MSK1/2 inhibitor. |
|
| Relations |
| BioSample |
SAMN02870113 |
| SRA |
SRX620771 |
