|
| Status |
Public on Mar 27, 2012 |
| Title |
2SKOA |
| Sample type |
RNA |
| |
|
| Source name |
Resting naïve CD4+ T cells (N) were purified to 95% purity from the spleens and lymph nodes of 4-6 week old BAF180 cKO Balb/c mice, (KO) using CD4+CD62L+ T cell Purification Kit II per manufacturer’s instructions (Miltenyi).
|
| Organism |
Mus musculus |
| Characteristics |
cell type: Stimulated Th2 cells
strain: Balb/c
genotype/variation: Pbrm1/BAF180 KO
|
| Treatment protocol |
Stimulated Th2 cells (2S) were prepared from resting Th2 cells by treatment with PMA (50ng/ml) and Ionomycin (500ng/ml) for 1.5 hours.
|
| Growth protocol |
Resting Th2 cells were prepared from resting naive cells by plating onto anti-CD3 (1ug/ml), anti-CD28 (2 ug/ml) coated plates at 1-2 x 106/ml in the presence of 10ng/ml IL-4, 10ug/ml anti-IFN-gamma. IL-2 (100U/ml) was added 24 hours later. Cultures were expanded in IL-2 (100U/ml) three days after initial culture.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated using RNeasy Kit (Qiagen). Quality and quantity of the total RNA was checked with the Agilent 2100 bioanalyzer using RNA 6000 Nano chips.
|
| Label |
Streptavidin-Cy3 bound to biotin-labeled cRNA.
|
| Label protocol |
standard Illumina protocol using 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 Sentrix MouseRef-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 labeled cRNA was detected by staining with streptavidin-Cy3.
|
| Scan protocol |
Arrays were scanned at a resolution of 0.8um using the Beadstation 500 X from Illumina.
|
| Description |
Stimulated Th2 cells, Pbrm1/BAF180 deficient, replicate A
|
| Data processing |
Data was extracted using the Illumina GenomeStudio software(v1.6.0). Any spots at or below the background intensity 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 below. Z-score = (raw value - avg)/std. Complete data including detection p-values will be included in the supplemental file.
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| |
|
| Submission date |
Aug 25, 2011 |
| Last update date |
Jun 22, 2020 |
| Contact name |
Supriyo De |
| Organization name |
NIA-IRP, NIH
|
| 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 |
| |
|
| Platform ID |
GPL6885 |
| Series (1) |
| GSE31676 |
IL-10 transcription is negatively regulated by BAF180, a component of the SWI/SNF chromatin remodeling enzyme |
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