Comment[ArrayExpressAccession] E-GEOD-58746 MAGE-TAB Version 1.1 Public Release Date 2014-08-12 Investigation Title Transcriptional response to stress in serum deprived mouse fibroblasts in the presence of MSK1/2 inhibitor. Comment[Submitted Name] Transcriptional response to stress in serum deprived mouse fibroblasts in the presence of MSK1/2 inhibitor. Experiment Description We have employed gene expression profiling in order to identify targets of transcriptional response to stress in resting mouse Swiss 3T3 fibroblasts, either untreated (control) or treated with anisomycin for 3 or 6 hours to induce the p38/MAP kinase pathway. In order determine transcriptional effects dependent on MSK1/2 kinase activity, H89 inhibitor was used in the study. Serum starved (72 h 0.2% FCS) mouse 3T3 cells were treated with anisomycin (188.5 nM) for 3 h or 6h (in duplicates) either with or without 15-min pre-treatment with MSK1/2 inhibitor H89 (10 uM). Untreated, serum-starved cells were used as a control. RNA was collected and gene expression profiling using strand-specific RNA-seq was performed. Term Source Name ArrayExpress EFO Term Source File http://www.ebi.ac.uk/arrayexpress/ http://www.ebi.ac.uk/efo/efo.owl Person Last Name Sawicka Sawicka Seiser Person First Name Anna Anna Christian Person Email anna.sawicka@mpibpc.mpg.de Person Affiliation Medical University of Vienna/MFPL Person Address Dep. of Medical Biochemistry, Medical University of Vienna/MFPL, Dr. Bohr-Gasse 9/2, Vienna, Austria Person Roles submitter Protocol Name P-GSE58746-4 P-GSE58746-1 P-GSE58746-3 P-GSE58746-2 Protocol Description 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 RefSeqand RPKM. 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). 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. 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). Protocol Type normalization data transformation protocol sample treatment protocol nucleic acid library construction protocol growth protocol Experimental Factor Name treatment Experimental Factor Type treatment Publication Title H3S28 phosphorylation is a hallmark of the transcriptional response to cellular stress. Publication Author List Sawicka A, Hartl D, Goiser M, Pusch O, Stocsits RR, Tamir IM, Mechtler K, Seiser C PubMed ID 25135956 Publication DOI 10.1101/gr.176255.114 Comment[SecondaryAccession] GSE58746 Comment[GEOReleaseDate] 2014-08-12 Comment[ArrayExpressSubmissionDate] 2014-06-23 Comment[GEOLastUpdateDate] 2014-11-24 Comment[AEExperimentType] RNA-seq of coding RNA Comment[SecondaryAccession] SRP043512 Comment[SequenceDataURI] http://www.ebi.ac.uk/ena/data/view/SRR1448825-SRR1448838 SDRF File E-GEOD-58746.sdrf.txt