Comment[ArrayExpressAccession] E-GEOD-42546 MAGE-TAB Version 1.1 Public Release Date 2014-04-09 Investigation Title Whole transcriptome analysis of postmortem human hippocampus dentate gyrus granlule cells Comment[Submitted Name] Whole transcriptome analysis of postmortem human hippocampus dentate gyrus granlule cells Experiment Description We investigated the transcriptome of dentate gyrus (DG) granule cells in postmortem hippocampus from 79 subjects with mental illness (schizophrenia, bipolar disorder, major depression) or non-psychiatric controls. Material for RNA-seq analysis was harvested from tissue slides using laser capture microdissection (LCM) and aRNA amplification. Equimolar amounts of triplicate aRNA samples for each of the 79 subjects were then pooled for the preparation of sequencing libraries. Sequencing libraries were prepared using Applied Biosystem's Total RNA Sequencing Kit, following the directions for Whole Transcriptome Libraries, and analyzed with an Applied Biosystems SOLiD 4 high-throughput sequencer. We compared the performance of different normalization methods (length normalization vs. noise reductin scaling), and assembled evidence for dysruption of signaling by the micro RNA miR-182 in subjects with major depression and schizophrenia. 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 Kohen Kohen Dobra Tracy Haugen Person First Name Ruth Ruth Adrian Julia Eric Person Mid Initials H Person Email ruko@uw.edu Person Affiliation University of Washington Person Address Psychiatry and Behavioral Sciences, University of Washington, 1959 Pacific Ave NE, Seattle, WA, USA Person Roles submitter Protocol Name P-GSE42546-2 P-GSE42546-1 Protocol Description Transcripts were mapped and counted using the Applied Biosystems software BioScope 1.2.1. Transcripts were mapped using the UCSC RefGene annotations and the BioScope default seed- and extend approach for mapping. Reads were mapped to the whole genome file, supplemented by a transcript annotation file allowing reads to align across known splice junctions with no gap penalty. Repeat and ambiguously mapped sequences were removed from the counts files using a UCSC RepeatMasker file. Only reads with a mapping quality of 10 or larger were used. The BEDTools bamToBed program was used to generate files of read locations. Only reads mapping to the opposite strand were counted. Genes expressing with greater than zero counts in four or more of our subjects (i.e. more than 5% of subjects) were excluded; for multiple transcripts at a given genomic location, e.g. due to the presence of splice variants, only one transcript with the highest number of counts was retained. Two alternate normalization methods, length normalization scaling, and noise reduction scaling, were used in parallel and their results compared. Genome_build: hg19 Supplementary_files_format_and_content: RawCounts, Raw counts per subject and mapped transcript, output of data processing step 3. Supplementary_files_format_and_content: CleanedRawCounts, Processed version of RawCounts file with genes expressing close to background and duplicate transcripts removed, output of data processing step 4. Supplementary_files_format_and_content: LengthNormalizationScaledCounts, Normalized and scaled counts, output of data processing step 5. Supplementary_files_format_and_content: NoiseReductionScaledCounts, Normalized and scaled counts, output of data processing step 5. Slide-mounted coronal cryostat sections from mid-hippocampus were stained and dehydrated using the Arcturus HistoGene LCM frozen section staining kit (Life Technologies, NY), and following the manufacturer’s instructions. From each subject, triplicate samples of about 2,000 DG granule cells each were harvested by laser capture microdissection, using an Arcturus AutoPix LCM system and CapSure Macro LCM caps (Molecular Devices, CA). Triplicates were processed separately during cell harvest, RNA extraction, and aRNA amplification in order to reduce experimental noise introduced during these stages of the experiment. Harvested cells were removed from the caps and RNA extracted using PicoPure RNA isolation kits (Life Technologies, NY). RNA was then linearly amplified over two rounds of aRNA amplification, using MessageAmp II aRNA amplification kits (Life Technologies, NY), and following the manufacturer’s protocol. The quality and concentration of aRNA was checked by spectrophotometry. Equimolar amounts of triplicate aRNA samples for each of the 79 subjects were then pooled for the preparation of sequencing libraries. Sequencing libraries were prepared using Applied Biosystem's Total RNA Sequencing Kit, following the directions for Whole Transcriptome Libraries, and analyzed with an Applied Biosystems SOLiD 4 high-throughput sequencer (Life Technologies, NY). Protocol Type normalization data transformation protocol nucleic acid library construction protocol Experimental Factor Name AGE AT DEATH DIAGNOSIS SEX Experimental Factor Type age at death diagnosis sex Comment[SecondaryAccession] GSE42546 Comment[GEOReleaseDate] 2014-04-09 Comment[ArrayExpressSubmissionDate] 2012-11-27 Comment[GEOLastUpdateDate] 2014-04-09 Comment[AEExperimentType] RNA-seq of coding RNA Comment[AdditionalFile:Data1] GSE42546_CleanedRawCounts.txt Comment[AdditionalFile:Data2] GSE42546_LengthNormalizedScaledCounts.txt Comment[AdditionalFile:Data3] GSE42546_NoiseReductionScaledCounts.txt Comment[AdditionalFile:Data4] GSE42546_RawCounts.txt Comment[SecondaryAccession] SRP017329 Comment[SequenceDataURI] http://www.ebi.ac.uk/ena/data/view/SRR620448-SRR620526 SDRF File E-GEOD-42546.sdrf.txt