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Sample GSM2221834 Query DataSets for GSM2221834
Status Public on Mar 22, 2017
Title 4M F2
Sample type SRA
 
Source name Hippocampus
Organism Mus musculus
Characteristics strain: C57BL/6J
Sex: Female
age: 4 Month
tissue: Hippocampus
Growth protocol 2 month old Male 5XFAD mice and female C57BL/6J mice were ordered from The Jackson Laboratory (Bar Harbor, ME). Hemizygous male transgenics and female WT animals were crossed yielding both transgenic and wild-type animals. Animals used in this study were all non-transgenic wild-types. Animals were group housed and kept on a 12 hour light-dark cycle. Food and water were provided ad libitum.
Extracted molecule total RNA
Extraction protocol The hippocampus (from both sides of the brain) was dissected and rapidly frozen in liquid nitrogen and stored at -80 degrees. Total RNA was extracted from the entire hippocampus from each animal using TRI Reagent (Sigma-Aldrich) according to manufacturer’s directions.
RNA from the dissected hippocampal tissue was extracted using a miRNeasy minikit (Qiagen, catalog #217004) with MaXtract high density columns (Qiagen, catalog #129056). A set of synthetic RNA standards were then added to 5 μg of RNA from each sample as spike-ins to assess the performance of each library (ERCC ExFold RNA Spike-In Mixes Ambion, catalog #4456739). mRNA was purified with a NEBNext mRNA magnetic isolation module (NEB, catalog #E7490L). cDNA libraries were generated from 50 ng of isolated mRNA using a NEBnext Ultra mRNA library preparation kit for Illumina sequencers (NEB, catalog #E7530L), and a unique 6-nucleotide index was incorporated to each library (NEB, catalog #E7335S, E7500S). The concentration of each cDNA library was estimated with qPCR using Illumina sequencing primers (KAPA Biosystems catalog # KK4835), and average fragment length was determined with a bioanalzyer high sensitivity DNA chip (Agilent Technologies, catalog #5076-4626). 12nM of each cDNA library was pooled into one of three cDNA library pools. Each of the three cDNA pools underwent additional quality control analysis via bioanalyzer and KAPA-PCR. 10pM of each cDNA library pool was sequenced on an Illumina HiSeq 2500
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina HiSeq 2500
 
Description sample name: 1057B
Data processing Sequences from Illumina adapters were trimmed using Trimmomatic (version 0.32) using the TruSeq3-SE.fa adapter sequence file provided with the program. A minimum length of 50 bases (MINLEN:50) was chosen as a cutoff for each read and 2 seed mismatches were allowed. All reads between 50 and 100 bases were included in further analyses.
The trimmed sequencing reads were aligned to the mouse genome (genome release GRCm38) using Tophat (2.0.13, --read-mismatches and --read-edit-dist set to 3). A General Transfer Format (GTF) file was used with Tophat (--GTF option) for the purpose of gene annotation (Mus_musculus.GRCm38.68.gtf).
Following mapping reads were futher processed (filtered, sorted and indexed) with Samtools and only reads that mapped to a single gene were used for further analysis.
The uniquely mapped reads were used to generate counts for each annotated gene using easyRNASeq (from Bioconductor version 3.0.2, geneModels summarization)
Differential expression analysis of count tables for female and male mice of various ages was performed using the default settings of DESeq2 (1.8.1, Benjamini-Hochberg FDR correction).
Cufflinks (2.1.1, --no-effective-length-correction, --max-bundle-length 10000000 --GTF Mus_musculus.GRCm38.68.gtf) was used to generate FPKM (fragments per kilobase per million reads) normalized values.
Genome_build: mm10 (GRCm38)
Supplementary_files_format_and_content: Microsoft Excel Worksheet (.xlsx). Contains unnormalized counts generated by easyRNASeq and normalized FPKM values generated by Cufflinks. The raw counts were used by DESeq2 for differential expression analysis.
 
Submission date Jun 30, 2016
Last update date May 15, 2019
Contact name Joseph Ligon Bundy
E-mail(s) [email protected]
Organization name Florida State University
Department Department of Biomedical Sciences
Lab Dr. Richard Nowakowski
Street address 1115 W Call St
City Tallahassee
State/province FL
ZIP/Postal code 32304
Country USA
 
Platform ID GPL17021
Series (1)
GSE83931 Sex differences in the molecular signature of the developing mouse hippocampus
Relations
BioSample SAMN05335324
SRA SRX1890434

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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