|
| Status |
Public on Dec 01, 2017 |
| Title |
CD34_LacZ_2 |
| Sample type |
RNA |
| |
|
| Source name |
human primary CD34 cells transduced with shLacZ control vector
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: primary CD34+ cells
|
| Treatment protocol |
Cells were lentivirally transduced with specific shvectors or a control shvector targeted against LacZ. Transduction was monitored by GFP, only cells with more than 95% GFP positiv cells were further processed. Cells were harvested 5 days after transduction.
|
| Growth protocol |
K562 cells were grown under standard conditions (ATCC). Human CD34+ cells were purified from mobilised apharesis samples with magnetic CD34+ antibody beads (Miltenyi). Cells were maintained and transduced in serum free expansion medium (StemSpan, StemCell Technologies)
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from cells using TRIzol® (Invitrogen/Life Technologies). mRNA was reverse-transcribed using oligo-dT and random hexameric primers and was quantified via qRT-PCR analysis using SYBR Green (Invitrogen/Life Technologies).
|
| Label |
cy3
|
| Label protocol |
Microarrays were done using the Low RNA Input linear Amplification Kit Plus, One Color protocol (Cat. N°: 5188-5339, 2007; Agilent Technologies, Inc., Santa Clara, CA) and the Agilent RNA Spike-In Kit for One color (Agilent Technologies, Inc. 2007; Cat. N°: 5188-5282) following the manufacturer's standard protocol. Global gene expression analysis was applied using the SurePrint G3 Human Gene Expression 8x60K v2 Microarray Kit, Product number G4851B, (Agilent Microarray Design ID 039494). 200 ng of total RNA were used as a starting material to prepare cDNA. cDNA synthesis and in vitro transcription (IVT) were performed according to the manufacturer's recommendation. Quantity and efficiency of the labeled amplified cRNA were determined using the NanoDrop ND-1000 UV-VIS Spectrophotometer version 3.2.1.
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| |
|
| Hybridization protocol |
The hybridizations were performed for 17 hours at 10 rpm and 65°C in the hybridization oven (Agilent). Washing and staining of the arrays were done according to the manufacturer's recommendation.
|
| Scan protocol |
Cy3 intensities were detected by one-color scanning using an Agilent DNA microarray scanner (G2505B) at 3 micron resolution. Scanned image files were visually inspected for artifacts and then analyzed.
|
| Data processing |
Intensity data were extracted using Agilent's Feature Extraction (FE) software (version 11.5.1.1) including a quality control based on internal controls using Agilent's protocol GE1_107_Sep09. All chips passed the quality control and were analyzed using the Limma packages of Bioconductor.
|
| |
|
| Submission date |
Dec 12, 2016 |
| Last update date |
Dec 01, 2017 |
| Contact name |
Gabriela Salinas |
| E-mail(s) |
[email protected]
|
| Organization name |
Universitaetsmedizin Goettingen
|
| Department |
Department of Pathology
|
| Lab |
NGS Integrative Genomics
|
| Street address |
Kreuzbergring 57
|
| City |
Goettingen |
| State/province |
Lower-Saxony |
| ZIP/Postal code |
37075 |
| Country |
Germany |
| |
|
| Platform ID |
GPL13497 |
| Series (1) |
| GSE92251 |
Gene expression alterations upon knock down of transcription factors |
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