|
| Status |
Public on Jun 24, 2011 |
| Title |
Th_WT-2 |
| Sample type |
RNA |
| |
|
| Source name |
Freshly isolated DP+SP4 thymocytes, control mice
|
| Organism |
Mus musculus |
| Characteristics |
strain/background: C57/BL6
genotype: Klf4fl/fl-CD4-Cre- (control)
tissue: thymus
cell type: DP+SP4 thymocytes
age: 8-10 weeks
|
| Treatment protocol |
Thymus were isolated from control mice and single-cell suspensions were prepared. CD4+ T cells were enriched with a CD4 T Cell Isolation Kit either from Miltenyi Biotec (Auburn, CA) or from Stem Cell Technologies (Vancouver, BC) according to the manufacturer's’ instructions. The purity of these isolated cells was >94%.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from the freshly isolated DP+SP4 thymocytes using the RNeasy Mini kit (Qiagen). The quality and quantity of total RNA were analyzed by the Agilent 2100 Bioanalyzer using RNA 6000 nano chips (Agilent Technologies, Palo Alto, CA).
|
| Label |
Biotin
|
| 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.
|
| |
|
| 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.
|
| Scan protocol |
Arrays were scanned at a resolution of 0.8um using the Beadstation 500 X from Illumina.
|
| Description |
Klf4fl/fl mice (Katz et al., 2002) were crossed with CD4-Cre mice (Lee et al., 2001) to generate Klf4fl/flCD4Cre+(KO) mice.
Freshly isolated DP+SP4 thymocytes from control mice.
|
| Data processing |
Data was extracted using the Illumina GenomeStudio software (v2010.1). Any spots at or below 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 below. Z-score = (raw value - avg)/std. Complete data including detection p-values is included in the data table.
|
| |
|
| Submission date |
Oct 21, 2010 |
| 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) |
| GSE24880 |
Krüppel-like factor 4 regulates T cell function |
|
