|
| Status |
Public on Jun 25, 2010 |
| Title |
Cord blood CD4+_control siRNA_Act_72h_rep2 |
| Sample type |
RNA |
| |
|
| Source name |
human cord blood CD4+ cell, nucleofected with control siRNA, activated, 72h
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: cord blood
cell type: CD4+ T cells
genome/variation: Control siRNA
biological replicate: 2
|
| Treatment protocol |
siRNA mediated knockdowns were perfomed using Nucleofector (Amaxa Biosystems) with a method described elsewhere (Tahvanainen et al. J Immunol Methods. 2006 Mar 20;310(1-2):30-9.). STAT6 siRNA #1 (5´- AAGCAGGAAGAACTGAAGTTT- 3`) or control siRNA (5´-GCGCGCTTTGTAGGATTCG-3´) was introduced into the cells. Cells were rested for 24 h and then activated throught the T cell receptor (plate bound antiCD3 500 ng/24-well, and soluble antiCD28 500 ng/ml, Immunotech, France). Th2 polarization was initiated with IL-4 (10ng/ml, R&D Systems). Cells were cultured in Yssel´s medium. At 48h, IL-2 was added to the cultures (17ng/ml, R&D Systems). Cells were harvested after 12, 24, 48 or 72 h of polarization.
|
| Growth protocol |
Umbilical cord blood was collected from healthy neonates born in Turku University Hospital, Hospital District of Southwest Finland. Mononuclear cells were isolated with Ficoll-Paque (Amersham Biosciences) gradient centrifugation, and CD4+ cell population was purified using magnetic beads (Dynal CD4 Positive Isolation Kit, Invitrogen) according to manufacturer's instructions.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted with RNeasy mini kit (Qiagen) according to manufacturer's instructions. In-column DNase (RNase-Free DNase Set, Qiagen) treatment was performed for 15 minutes. RNA was eluted in dH2O.
|
| Label |
biotin
|
| Label protocol |
Amplification was started using 100ng total RNA with Ambion's Illumina RNA TotalPrep Amplification kit (cat no AMIL1791). IVT reaction took overnight (14h) and during it cRNA was biotinylated. Both before and after the amplifications the RNA/cRNA concentrations where checked with Nanodrop ND-1000 and RNA/cRNA quality was controlled by BioRad’s Experion electrophoresis station.
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| |
|
| Hybridization protocol |
1.5 ug each sample was hybridized to Illumina’s Sentrix HumanWG-6 Expression BeadChips, version 2 (cat no BD-25-112) at 55C overnight (18½ h) according to Illumina Whole-Genome Gene Expression Direct Hybridization protocol, revision A. Hybridization was detected with 1 ug/ml Cyanine3-streptavidin, Amersham Biosciences (cat no 146065).
|
| Scan protocol |
Chips were scanned with Illumina BeadArray Reader. The numerical results were extracted with GenomeStudio v1.0 without any normalization.
|
| Description |
Control_Act_72h_rep2
|
| Data processing |
The data were quantile-normalized using the R/Bioconductor package affy and log2-transformed.
|
| |
|
| Submission date |
Aug 27, 2009 |
| Last update date |
Jun 09, 2010 |
| Contact name |
Henna Kallionpää |
| E-mail(s) |
henna.kallionpaa@btk.fi
|
| Phone |
+358-2-333-8001
|
| Organization name |
University of Turku
|
| Department |
Turku Centre for Biotechnology
|
| Lab |
Riitta Lahesmaa
|
| Street address |
P.O. Box 123
|
| City |
Turku |
| ZIP/Postal code |
FIN-20521 |
| Country |
Finland |
| |
|
| Platform ID |
GPL6102 |
| Series (2) |
| GSE17851 |
Genome-wide analysis of STAT6 target genes in IL-4 treated human cord blood CD4+ cells |
| GSE18017 |
Stat6 mediated regulation of transcription to initiate Th2 program in human T cells |
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