tissue: whole brain strain: C57BL/6J (B6) gender: Male
Extracted molecule
total RNA
Extraction protocol
Naïve, adult, male C57BL/6J (B6) (n=12) and DBA/2J (D2) (n=12) strain mice were euthanized by cervical dislocation, the whole brain was rapidly removed and flash frozen in liquid nitrogen. Total RNA was isolated using TRIzol® reagent (Invitrogen, Carlsbad, CA) in a one-step guanidine isothiocyanate procedure. For microarray analyses, the extracted RNA was purified using RNeasy (Qiagen, Valencia, CA). RNA samples were evaluated by ultraviolet spectroscopy for purity and concentration.
Label
Biotin
Label protocol
Samples containing at least 10 μg of total RNA were sent to the Oregon Health & Science University Gene Microarray Shared Resource facility for further quality assessment using an Agilent 2100 BioAnalyzer and for GeneChip array analysis. Because two samples were scanned by a different scanner , the dataset reported here includes whole brain samples from 22 individual mice (10 B6 and 12 D2). After ribosomal RNA reduction, amplification and labeling, whole-brain total RNA samples were each hybridized to AffymetrixGeneChip® Mouse Exon 1.0 ST Array (24 arrays total). The procedures used follow the Affymetrix Whole Transcript Sense Target Labeling Assay, rev3, protocol. Additional details can be found at (https://www.affymetrix.com/support/downloads/manuals/wt_sensetarget_label_manual.pdf).
Hybridization protocol
Samples containing at least 10 μg of total RNA were sent to the Oregon Health & Science University Gene Microarray Shared Resource facility for further quality assessment using an Agilent 2100 BioAnalyzer and for GeneChip array analysis. Because two samples were scanned by a different scanner , the dataset reported here includes whole brain samples from 22 individual mice (10 B6 and 12 D2). After ribosomal RNA reduction, amplification and labeling, whole-brain total RNA samples were each hybridized to AffymetrixGeneChip® Mouse Exon 1.0 ST Array (24 arrays total). The procedures used follow the Affymetrix Whole Transcript Sense Target Labeling Assay, rev3, protocol. Additional details can be found at (https://www.affymetrix.com/support/downloads/manuals/wt_sensetarget_label_manual.pdf).
Scan protocol
Standard Affymetrix protocol.
Description
14B-11H1A_B05_334RH.CEL
Data processing
All CEL files were processed using the Affy package (version 1.12.2) in the statistical programming environment R under the Bioconductor package [R version 2.6.0 (http://www.r-project.org), Bioconductor2.1 (http://www.bioconductor.org)]. All data was RMA [11] background corrected and normalized. Data were summarized at the probeset level using the median polish. The Mouseexonpmcdf package was utilized for summarization at the probeset-level (http://xmap.picr.man.ac.uk/download/). The data were SNP masked at the probe level. Prior to the summarization step at the probeset level described above, individual probes were masked (expression value replaced by 'null') if there was a known SNP within the boundaries of the probe. The SNP mask was built by comparing the Affymetrix design time annotation files (GFF) files (based on NCBI mouse build 36) for each chromosome to known SNP locations in DBSNP. A total of 12,101 probesets were masked to some degree, representing 5.4% of the core probesets.
