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Sample GSM1044853 Query DataSets for GSM1044853
Status Public on Apr 09, 2014
Title D6
Sample type SRA
 
Source name postmortem hippocampus DG granule cells
Organism Homo sapiens
Characteristics age at death: 42
gender: M
diagnosis: depression
Extracted molecule total RNA
Extraction protocol 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).
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model AB SOLiD 4 System
 
Description LCM harvest + two rounds of aRNA amplification
Data processing 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.
 
Submission date Nov 27, 2012
Last update date May 15, 2019
Contact name Ruth Kohen
E-mail(s) ruko@uw.edu
Organization name University of Washington
Department Psychiatry and Behavioral Sciences
Street address 1959 Pacific Ave NE
City Seattle
State/province WA
ZIP/Postal code 98195-6560
Country USA
 
Platform ID GPL13393
Series (1)
GSE42546 Whole transcriptome analysis of postmortem human hippocampus dentate gyrus granlule cells
Relations
SRA SRX206654
BioSample SAMN01820027

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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