Comment[ArrayExpressAccession] E-GEOD-60752 MAGE-TAB Version 1.1 Public Release Date 2015-04-10 Investigation Title Time resolved ribosome profiling study of oxygen and glucose deprivation of rat pheochromocytoma cells Comment[Submitted Name] Time resolved ribosome profiling study of oxygen and glucose deprivation of rat pheochromocytoma cells Experiment Description Oxygen and glucose metabolism plays a pivotal role in many (patho)physiological conditions. In particular, oxygen and glucose deprivation (OGD) occurs during ischemia and stroke, resulting in extensive tissue injury and cell death. We applied time-resolved ribosome profiling technique to assess early events at the level of gene expression in rat pheochromocytoma PC12 cells during short-term OGD. Most substantial alterations in transcripts levels and their translation were seen to occur in the first 20 minutes of OGD. The rapid adaptation of translation apparatus to OGD is global and involves altered elongation and initiation rates. We also observed salient and reproducible alterations in ribosome densities of individual mRNAs such as increased translation of particular upstream Open Reading Frames (uORFs); induced site-specific arrests of the ribosomes and synthesis of extended protein isoforms. Ribosome profiling (with mRNA-seq sequencing) was carried out at 0,20,40 and 60 minutes of OGD. Two biological replicates were used. 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 Baranov Andreev O’Connor Zhdanov Dmitriev Shatsky Papkovsky Baranov Person First Name Pavel Dmitry Patrick Alexander Ruslan Ivan Dmitry Pavel Person Mid Initials V E B V I N B V Person Email p.baranov@ucc.ie Person Affiliation University College Cork Person Phone +353 420 5419 Person Address Biochemistry and Cell Biology, University College Cork, Western Gateway Building, UCC, Cork, Co. Cork, Ireland Person Roles submitter Protocol Name P-GSE60752-2 P-GSE60752-1 Protocol Description Initial processing; Cutadapt was used for the removal of the adapter sequence (CTGTAGGCACCATCAATAGATCGGAAGAGCACACGTCTGAACTCCAGTCA). The reads were then aligned to rRNA and the introduced “spike-in” (ATGTACACGGAGTCGACCCGCAACGCGA) to remove reads that originated from these sources. Reads were aligned to the rat RefSeq gene catalogue downloaded from NCBI on January 2014 (ftp://ftp.ncbi.nih.gov/refseq/R_norvegicus/mRNA_Prot/) with bowtie. The alignment parameters used where (-a -m 100 -v 2 –norc). Differential expression analysis; We calculated the number of reads aligning to any of Refseq transcripts originating from the same gene. Ribo-seq reads were assigned to mRNA coordinates based on the inferred location of the A-site. Those of length less than 29 or greater than 35 were discarded. For genes that had transcripts with annotated coding regions we used only ribo-seq reads aligning to them, otherwise those mapped to the entire transcript were included. Some ribo-seq reads were mapped to more than 1 region of a transcript. We assigned them to unique locations based on the following priority order: annotated coding region, 5' leader, 3' UTR. We included ambiguously aligned reads that mapped to less than 4 locations, for these reads the value of aligned reads was reduced by the number of its alignment locations. To reduce the effects of misalignment on a genes estimated level of expression we excluded coordinates corresponding to the three highest density peaks of the ribosome density from each transcript. Further normalisation to remove the differences due to the total number of mapped reads was carried out. The read counts aligning to a feature in the sample k were multiplied by the rescaling factor x[k]/min(x[i]) where x[k] is the number of all sequence reads aligning to all features within the sample k, and min(x[i]) is the number of all reads aligning to all features in the sample i which has the smallest number of reads. This rescaling was carried out independently for the mRNA-seq and ribo-seq data, however both replicates were normalised together to allow for comparison between replicates. To identify differentially expressed genes we carried out the Z-score transformation of log ratios in read counts by calculating local Z-scores for the groups of 300 genes clustered based on the lowest signal of their expression mRNA-seq, ribo-seq or both depending on what is being compared. The gene was classified as upregulated if (Z1+Z2)/2 > T and downregulated if (Z1+Z2)/2 < -T, where Z1 and Z2 are Z-scores for the same gene obtained in two replicas and T is the threshold. The threshold T was set based on the desired false discovery rate (FDR). The number of false positives was estimated as the number of genes for which (|Z1|+|Z2|)/2>T when Z1Z2<0. A gene was considered robustly regulated only if it was detected as regulated after exclusion of coordinates corresponding to three highest density peaks of the ribosome density for each of its transcripts. Genome_build: rat RefSeq gene catalogue downloaded from NCBI (mRNA_Prot/) Supplementary_files_format_and_content: The tab delimited csv files record the number of mapped reads that aligned to the gene in the RefSeq catalogue after the alignment step. Each row contains the record of a Gene with first 3 columns indicating its Gene symbol, its annotation classification (coding, ncRNA, rRNA,transcript,pseudo,linc) and the number of alignments. Three more columns are used for coding transcripts. These columns indicates the number of alignments to the 5'leader, the acORF and the 3'UTR. The supplmentary_table.xlsx is available on the series record and containsgenes found to differentially regulated as well as records differential gene expression analysis of the dataset. Detergent lysis was performed in the dish, and after DNAse treatment sample was taken for mRNA isolation. Lysates were subjected to ribosome footprinting by RNAse I treatment. After sucrose density gradient fractionation ribosome-protected fragments were purified from 80S peak fractions. In parallel, mRNA isolated with Oligotex kit (Quiagen) was subjected to alkaline hydrolysis and fragments of the same size as ribosome protected fragments were isolated. Library preparation was carried out as described in Ingolia et al (2012),Nature Protocols Protocol Type normalization data transformation protocol nucleic acid library construction protocol Experimental Factor Name treatment rna type Experimental Factor Type treatment rna type Publication Title Oxygen and glucose deprivation induces widespread alterations in mRNA translation within 20 minutes. Publication Author List Andreev DE, O'Connor PB, Zhdanov AV, Dmitriev RI, Shatsky IN, Papkovsky DB, Baranov PV PubMed ID 25943107 Publication DOI 10.1186/s13059-015-0651-z Comment[SecondaryAccession] GSE60752 Comment[GEOReleaseDate] 2015-04-10 Comment[ArrayExpressSubmissionDate] 2014-08-26 Comment[GEOLastUpdateDate] 2015-06-16 Comment[AEExperimentType] RNA-seq of coding RNA Comment[AdditionalFile:Data1] GSE60752_supplementary_data.xls Comment[SecondaryAccession] SRP045777 Comment[SequenceDataURI] http://www.ebi.ac.uk/ena/data/view/SRR1557705-SRR1557720 SDRF File E-GEOD-60752.sdrf.txt