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Sample GSM2551901

Query DataSets for GSM2551901

Status

Public on Feb 13, 2019

Title

1M M5 AD

Sample type

SRA

Source name

Hippocampus

Organism

Mus musculus

Characteristics

strain: B6.Cg-Tg(APPSwFlLon,PSEN1*M146L*L286V)6799Vas/Mmjax
Sex: Male
age: 1 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 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

1071W

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.
Supplementary_files_format_and_content: Supplemental_file_1_genelists.xlsx: Multi-tab Excel file containing summary statistics from various pairwise comparisons of gene expression performed by DESeq2.
Supplementary_files_format_and_content: Supplemental_file_2_QPCR_validation.xlsx: Multi-tab Excel file containing a report of a qRTPCR experiment performed on a subset of these samples for validation purposes.

Submission date

Mar 28, 2017

Last update date

May 15, 2019

Contact name

Joseph Ligon Bundy

E-mail(s)

josephlbundy@gmail.com

Organization name

Florida State University

Department

Department of Biomedical Sciences