|
| Status |
Public on Aug 15, 2015 |
| Title |
R2t0 |
| Sample type |
RNA |
| |
|
| Source name |
HeLa cell line,0hr stress
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HeLa
cell type: cervical cancer cell line
exposed to: none (untreated control)
|
| Treatment protocol |
DTT was used to stress the cells for different lengths, 0, 0.5, 1, 2, 8, 16, 24, 30hrs. To account for different cellular ages at harvest, the experiment was conducted “backwards”. The cells were stressed at different time points 0, 1, … 30hrs before collection and all the stress-end points were aligned at one time point.
|
| Growth protocol |
HeLa cells were used to conduct the 30 hour-long time course experiment. The cells were collected simultaneously at the end point. By this way, all cells grew for the same time period, and the changes of RNA are only due to the effects of stress. The cultures were incubated at 37 ºC in a humidified incubator with 5% CO2.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA extraction was conducted using Trizol (Sigma) followed by the PhaseLock Gel-Heavy manufacturer protocol (5-Prime) and finally the RNA was purified using the RNA MinElute Kit (QIAGEN). Nanodrop ND-1000 was used to quantify RNA and assess quality.
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 50 ng RNA using the One-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Protocol (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
|
| |
|
| Hybridization protocol |
600 ng of Cy3-labelled cRNA was fragmented at 60°C for 30 minutes in a reaction volume of 25 ul containing 1x Agilent fragmentation buffer and 2.5x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 25 ul of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Whole Human Genome Oligo Microarrays (G4112A) for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent), 1 minute with 37°C GE Wash buffer 2 (Agilent), and 10 seconds in acetonitrile.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2565CA) using one color scan setting for 8x60k array slides (Scan Area 61x21.6 mm, Scan resolution 3um).
|
| Description |
Gene expression in control without DTT induced stress in HeLa cells
US93503718_252800415820_S01_GE1_107_Sep09_2_1
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 10.7.3.1 (Agilent) using default parameters (protocol GE1_107_Sep09 and Grid: 028004_D_F_20110819).
The normalized data were further processed using ENSG names instead of probe names (provided in the 'normalized_ENSG_IDs.txt', 'normalized_ENSG_IDs_log_filtered_LOWESSsmoothed.txt' files). The probe IDs were converted to transcript IDs, and then to Gene ENSG IDs based on ensembl_mart_db from biomaRt by R programming software.
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| |
|
| Submission date |
Apr 15, 2015 |
| Last update date |
Aug 15, 2015 |
| Contact name |
Zhe Cheng |
| E-mail(s) |
zhecheng2010@gmail.com
|
| Phone |
2129983658
|
| Organization name |
New York University
|
| Department |
Bilogy Department
|
| Lab |
Christine Vogel lab
|
| Street address |
12 Waverly Place, 4th Floor
|
| City |
New York |
| State/province |
New York |
| ZIP/Postal code |
10003 |
| Country |
USA |
| |
|
| Platform ID |
GPL13607 |
| Series (1) |
| GSE67901 |
Expression dynamics in mammalian cells responding to protein misfolding stress |
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