|
| Status |
Public on Oct 01, 2018 |
| Title |
HEK293T_139CWC27_alt-splice_rep3 |
| Sample type |
RNA |
| |
|
| Source name |
HEK293T, 139CWC27, rep3
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HEK293T
|
| Treatment protocol |
Cells were washed with 1X PBS before freezing in preparation for RNA extraction.
|
| Growth protocol |
HEK293T cells were transduced with titered lentivirus containing either shRNA from the MISSION library or with scrambled shRNA. Puromycin was used to select for incorporation. Cells were grown to 80% confluency and harvested.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
total RNA was extracted and purified using the Bioline ISOLATE II kit according to manufacturer's instructions.
|
| Label |
biotin
|
| Label protocol |
Labeling was conducted by the Molecular Profiling Facility at the University of Pennsylvania. All protocols were conducted as described in the Affymetrix WT Plus Reagent Kit Manual and the Affymetrix GeneChip Expression Analysis Technical Manual. Briefly, 250ng of total RNA was converted to first-strand cDNA using reverse transcriptase primed by poly(T) and random oligomers that incorporated the T7 promoter sequence. Second-strand cDNA synthesis was followed by in vitro transcription with T7 RNA polymerase for linear amplification of each transcript, and the resulting cRNA was converted to cDNA, fragmented, assessed by Bioanalyzer, and biotinylated by terminal transferase end labeling.
|
| |
|
| Hybridization protocol |
Hybridization was conducted by the Molecular Profiling Facility at the University of Pennsylvania. Five and a half micrograms of labeled cDNA were added to Affymetrix hybridization cocktails, heated at 99ºC for 5 min and hybridized for 16 h at 45ºC to Human Transcriptome 2.0 ST GeneChips (Affymetrix Inc., Santa Clara CA) using the GeneChip Hybridization oven 645. The microarrays were then washed at low (6X SSPE) and high (100mM MES, 0.1M NaCl) stringency and stained with streptavidin-phycoerythrin. Fluorescence was amplified by adding biotinylated anti-streptavidin and an additional aliquot of streptavidin-phycoerythrin stain.
|
| Scan protocol |
Scanning was conducted by the Molecular Profiling Facility at the University of Pennsylvania. A GeneChip 3000 7G scanner was used to collect fluorescence signal.
|
| Data processing |
Probeset and gene normalized data was calculated from cel files in Affymetrix Expression Console software using RMA with default parameters.
|
| |
|
| Submission date |
Jul 16, 2018 |
| Last update date |
Oct 01, 2018 |
| Contact name |
Tara Davis |
| E-mail(s) |
Tara.Davis@DrexelMed.edu
|
| Organization name |
Drexel University College of Medicine
|
| Department |
Biochemistry and Molecular Biology
|
| Lab |
Lab 10127
|
| Street address |
245 N. 15th St. MS 497
|
| City |
Philadelphia |
| State/province |
PA |
| ZIP/Postal code |
19102 |
| Country |
USA |
| |
|
| Platform ID |
GPL17585 |
| Series (1) |
| GSE117144 |
Comparison of CWC27 knockdown in HEK293T cells compared to scrambled shRNA control |
|
| Relations |
| Alternative to |
GSM3272156 |
