|
| Status |
Public on Feb 19, 2014 |
| Title |
HCT116 H2B-GFP 5/4_R3 |
| Sample type |
RNA |
| |
|
| Source name |
HCT116 cell line, tetrasomic for Chr. 5, replicate 3
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: human colon carcinoma cell line
variation: 4x Chr. 5
cell line: HCT116
|
| Treatment protocol |
No treatment
|
| Growth protocol |
Cell lines were cultered in DMEM with 10% FBS at 5% CO2, 37°C
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extraction with the Quiagen mRNeasy Mini Kit
|
| Label |
Cy3
|
| Label protocol |
500 ng (HCT116 3/3 and HCT116 H2B-GFP 5/4) or 100 ng total RNA per sample were introduced into an RT-IVT reaction. Prior to RT-IVT, the total RNA samples were spiked with in-vitro synthesized polyadenylated transcripts (One-Color RNA Spike-In Mix, Agilent Technologies) which serve as an internal labeling control for linearity, sensitivity and accuracy. The spiked total RNA was reverse transcribed into cDNA and then converted into labeled cRNA by in-vitro transcription (HCT116 3/3 and HCT116 H2B-GFP 5/4: Quick-Amp Labeling Kit One-Color, Agilent Technologies; all others: Low Input Quick-Amp Labeling Kit One-Color, Agilent Technologies) incorporating Cyanine-3-CTP according to the manufacturer´s instructions.
|
| |
|
| Hybridization protocol |
Following cRNA clean-up and quantification (NanoDrop ND-1000 Spectrophotometer, peqlab) 1.65 µg (HCT116 3/3 and HCT116 H2B-GFP 5/4) or 0.60 µg (others) of each Cyanin-3-labeled cRNA sample was fragmented and prepared for One-Color based hybridization (Gene Expression Hybridization Kit, Agilent). Each cRNA sample was hybridized at 65 °C for 17 hrs on separate Whole Human Genome Oligo Microarrays (4x44K format) (for HCT116 3/3 and HCT116 H2B-GFP 5/4) or SurePrint G3 Human Gene Expression Arrays (8x60K).
|
| Scan protocol |
Fluorescent signal intensities were detected with Scan Control 8.1.3. Software (Agilent Technologies) (HCT116 3/3 and HCT116 H2B-GFP 5/4) or Scan Control A.8.4.1. Software (Agilent Technologie) on the Agilent DNA Microarray Scanner.
|
| Data processing |
Signal intensities were extracted from the image using Feature Extraction 10.5.1 Software (Agilent Technologies) and the design file 014850_D_F_20090416.xml (HCT116 3/3 and HCT116 H2B-GFP 5/4) or 10.7.3.1 Software and the design file 028004_D_F_20120130.xml (others). Data was background subtracted; log2 transformed and normalized. Medians of replicates were calculated and a ratio of the aneuploid cell line (median) vs the corresponding WT cell line (median) was used for further analysis (indicated in the column header).
|
| |
|
| Submission date |
Jun 11, 2013 |
| Last update date |
Feb 19, 2014 |
| Contact name |
Milena Dürrbaum |
| Organization name |
Max Planck Institute of Biochemistry
|
| Department |
Maintenance of Genome Stability
|
| Street address |
Am Klopferspitz 18
|
| City |
Martinsried |
| ZIP/Postal code |
82152 |
| Country |
Germany |
| |
|
| Platform ID |
GPL4133 |
| Series (1) |
| GSE47830 |
Uniform response to aneuploidy in human cells |
|
