|
| Status |
Public on Aug 13, 2015 |
| Title |
Donor_5; 1uM SAHA |
| Sample type |
RNA |
| |
|
| Source name |
CD4+ T cells; 1uM SAHA
|
| Organism |
Homo sapiens |
| Characteristics |
individual: Donor 5
tissue: peripheral blood
cell type: CD4+ T cells
agent: SAHA
dose: 1uM
rin: 8.5
|
| Treatment protocol |
CD4+ T cells were counted and aliquoted into 6-well plates; 5 million cells per well at a concentration of 2.5 million cells per mL of media (RPMI 1640 with 5% human serum AB). Wells containing CD4+ T cells were treated with SAHA (Merck) at 0.34 μM, 1 μM, 3 μM, or 10 μM in 0.1% DMSO, or with only 0.1% DMSO. Treated cells were incubated at 37°C, 5% CO2 for 24 hours.
|
| Growth protocol |
Human peripheral blood was drawn according to institutional review board approved protocols from 9 healthy HIV-seronegative donors by venipuncture and collected into sodium heparin containing tubes. Peripheral blood mononuclear cells (PBMCs) were collected from fresh whole blood by centrifugation. Primary CD4+ T cells were isolated from PBMCs with Rosette Sep CD4+ Isolation Kits according to the manufacturers protocol (Stem Cell Technologies, Inc.). CD4+ T cells were incubated at 37°C, 5% CO2 overnight in fresh media (5% human serum AB in RPMI 1640). CD4+ T cell purity and activation were assessed with flow cytometry. All CD4+ T cell samples included in microarray analysis had >95% purity with fewer than 10% activated cells (i.e., <10% expressing HLA-DR).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted from treated cells using Qiagen RNeasy RNA isolation kits according to manufacturer’s protocols. RNA integrity numbers (RINs) were determined with the Agilent 2100 Bioanalyzer (Agilent Technologies, Inc). RNA samples submitted for microarray analysis (N=6) had RINs between 7.9 and 9.0 with an average RIN of 8.5. cRNA was generated and hybridized to IlluminaHT-12 v3 BeadChips (48,803 probes).
|
| Label |
cy3
|
| Label protocol |
Biotinylated cRNA were prepared by Asuragen, Inc using standard Illumina protocols
|
| |
|
| Hybridization protocol |
Standard Illumina hybridization protocol; conducted by Asuragen, Inc
|
| Scan protocol |
Standard Illumina scanning protocol; conducted by Asuragen, Inc.
|
| Description |
Sample name: 5590177014_E
Donor_5
|
| Data processing |
Expression data were extracted with GenomeStudio software (Illumina); genes with undetectable expression were excluded from analysis. The lumi package [12] in bioconductor was used to transform (variance-stabilizing) and normalize (robust spline) the raw expression data. Genes significantly modulated across SAHA doses were identified using a likelihood ratio test (LRT) in the R package Isogene GX [13]. The family wise error rate was controlled using Bonferroni-adjusted p-values (p < 0.05).
|
| |
|
| Submission date |
Mar 17, 2015 |
| Last update date |
Aug 13, 2015 |
| Contact name |
Brian Reardon |
| Organization name |
UCSD
|
| Department |
Medicine
|
| Street address |
9500 Gilman Drive
|
| City |
La Jolla |
| State/province |
CA |
| ZIP/Postal code |
92093 |
| Country |
USA |
| |
|
| Platform ID |
GPL6947 |
| Series (1) |
| GSE66994 |
Dose-Responsive Gene Expression in Suberoylanilide Hydroxamic Acid (SAHA) Treated Resting CD4+ T Cells |
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