|
Status |
Public on Jul 14, 2016 |
Title |
Uninfected HeLa cells |
Sample type |
RNA |
|
|
Source name |
Unifected HeLa cells transduced with shRNA library and cultured for 17 days
|
Organism |
Homo sapiens |
Characteristics |
cell type: Human epithelial carcinoma cell line cell line: HeLa infection: Uninfected
|
Treatment protocol |
Latently infected and unifected HeLa cells were transduced with the lentiviral shRNA library for 6 hours at an MOI of approximately 0.4. Transduced cells were selected with Puromycin and cultured for a total of 17 days, which was experimentally determined to provide ample time for shRNA expression and knockdown of the target gene, as well as viral reactivation and viral protein-mediated cell death in the experimental sample. In other words, a negative selection protocol was used to identify shRNAs that activated latent virus by taking advantage of the cytotoxic effects of the HIV-1 viral protein Vpr.
|
Growth protocol |
HeLa cells transduced with the shRNA library were cultured for 17 days in standard conditions (37 degrees celsius with 5% CO2) in MEM GlutaMAX medium (Life Technologies, Grand Island, NY) supplemented with 10% FetalClone III Serum (Thermo Scientific Hyclone, Logan, UT), 2X MEM Non-essential amino acids solution (Life Technologies, Grand Island, NY), 100U/mL penicillin-100μg/mL streptomycin solution (Life Technologies, Grand Island, NY), and 0.75μg/mL Puromycin (Sigma-Aldrich, St. Louis, MO).
|
Extracted molecule |
total RNA |
Extraction protocol |
System Biosciences isolated total RNA, reverse transcribed the RNA into cDNA, and amplified the cDNA as a fee for service according to GeneChip Expression Analysis Technical Manual (P/N 702232 Rev. 3): Chapter 2
|
Label |
Biotin
|
Label protocol |
System Biosciences amplified the cDNA using Biotin-conjugated primers as a fee for service according to GeneChip Expression Analysis Technical Manual (P/N 702232 Rev. 3): Chapter 2
|
|
|
Hybridization protocol |
System Biosciences hybridized the labeled cDNA to the Affymetrix GeneChip Array (HG-U133+ 2.0) using GeneChip Hybridization Oven 640 and washed and stained using GeneChip Fluidics Station 450 as a fee for service according to GeneChip Expression Analysis Technical Manual (P/N 702232 Rev. 3): Chapter 3
|
Scan protocol |
GeneChips were scanned using GeneChip Scanner 3000 7G
|
Description |
Uninfected
|
Data processing |
The data were analyzed with GeneChip Operating Software v1.4 using default analysis and normalization settings GeneChip Operating Software v1.4 signal intensities per probeset ID were averaged for each individual microarray. In other words, probeset signal intensities were averaged for the experimental sample microarray and probeset signal intensities were averaged for the reference control sample microarray). Averages per probeset ID are reported in two separate columns in the matrix file according to which array they were obtained from.
|
|
|
Submission date |
Jul 25, 2014 |
Last update date |
Jul 14, 2016 |
Contact name |
Joseph Dougherty |
E-mail(s) |
doughejp@rwjms.rutgers.edu
|
Phone |
732-235-4588
|
Organization name |
Rutgers University - Robert Wood Johnson Medical School
|
Department |
Department of Pharmacology
|
Lab |
Research Towers Rm. 813
|
Street address |
675 Hoes Lane West
|
City |
Piscataway |
State/province |
NJ |
ZIP/Postal code |
08854 |
Country |
USA |
|
|
Platform ID |
GPL570 |
Series (1) |
GSE59758 |
Genome-wide shRNA screen to identify cellular regulators of the maintenance of HIV-1 latency |
|