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Status |
Public on Dec 22, 2014 |
Title |
WT-SA_12_HR-1 |
Sample type |
RNA |
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Source name |
microglia, wild type control, 12hr
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Organism |
Mus musculus |
Characteristics |
strain: C57BL/6 genotype: wild type developmental stage: neonatal age: 2-4 days tissue: cerebral cortex cell type: microglia from primary mixed glial culture treatment: Staphylococcus aureus stimulation time: 12 hours
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Biomaterial provider |
Mice from Jackson Laboratories, Bar Harbor, ME.
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Treatment protocol |
A USA300 community-acquired methicillin-resistant S. aureus (CA-MRSA) clinical isolate recovered from a patient with a fatal brain abscess was kindly provided by Dr. Costi Sifri (University of Virginia School of Medicine, Charlottesville). Bacterial strains were heat-inactivated and used to stimulate WT microglia at 107 colony forming units (cfu)/well for 6 or 12 hours.
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Growth protocol |
Primary mixed glial cultures were prepared on the same day from the cerebral corticies of neonatal WT mice (2-4 days of age). Microglia were harvested from mixed glial cultures using a differential shaking technique with a purity of >98%.
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Extracted molecule |
total RNA |
Extraction protocol |
Total RNA was extracted from the stimulated microglia using Trizol (Invitrogen). The quality and quantity of total RNA were analyzed by the Agilent 2100 Bioanalyzer using RNA 6000 nano chips (Agilent Technologies, Palo Alto, CA).
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Label |
biotin
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Label protocol |
Standard Illumina protocol using the Illumina TotalPrep RNA Amplification Kit (Ambion; Austin, TX, cat # IL1791). In short, 0.5g of total RNA was first converted into single-stranded cDNA with reverse transcriptase using an oligo-dT primer containing the T7 RNA polymerase promoter site and then copied to produce double-stranded cDNA molecules. The double-stranded cDNA was cleaned and concentrated with the supplied columns and used in an overnight in-vitro transcription reaction where single-stranded RNA (cRNA) was generated and labeled by incorporation of biotin-16-UTP.
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Hybridization protocol |
Standard Illumina protocol. In short, a total of 0.75ug of biotin-labeled cRNA was hybridized at 58 degrees C for 16 hours to Illumina's SentrixMouse Ref-8, v2 Expression BeadChips (Illumina, San Diego, CA). Each BeadChip has 24,000 well-annotated RefSeq transcripts with approximately 30-fold redundancy. The arrays were washed, blocked and the biotin-labeled cRNA was detected by staining with streptavidin-Cy3.
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Scan protocol |
Arrays were scanned at a resolution of 0.8um using the Beadstation 500 X from Illumina.
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Description |
RNA from the S. aureus-stimulated microglia (12 hr) of WT C57BL/6 mice.
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Data processing |
Data was extracted using the Illumina BeadStudio software (v3.4). Any spots at or in the Sample data table. the background were filtered out using an Illumina detection p value of 0.02 and above. The natural log of all remaining scores were used to find the avg and std of each array and the z-score normalization was calculated and presented in the Sample data table. Z-score = (raw value - avg)/std. Complete data including detection p-values is included in the supplementary file that is linked to the Series record.
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Submission date |
Oct 26, 2010 |
Last update date |
Jun 22, 2020 |
Contact name |
Supriyo De |
Organization name |
NIA-IRP, NIH
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Department |
Laboratory of Genetics and Genomics
|
Lab |
Computational Biology & Genomics Core
|
Street address |
251 Bayview Blvd
|
City |
Baltimore |
State/province |
Maryland |
ZIP/Postal code |
21224 |
Country |
USA |
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Platform ID |
GPL6885 |
Series (1) |
GSE24935 |
Toll-like receptor 2 (TLR2)-TLR9 crosstalk dictates IL-12 family cytokine production in microglia |
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