|
| Status |
Public on Apr 30, 2016 |
| Title |
YAP_Knockdown_rep2 |
| Sample type |
SRA |
| |
|
| Source name |
Nf2-null Schwann cells treated with YAP pooled siRNA
|
| Organism |
Mus musculus |
| Characteristics |
tissue: Nf2-null schwannoma
cell line: SC4 Schwann cells
treatment: YAP Smartpool siRNA (GE Life Sciences)
|
| Biomaterial provider |
Dr. Helen Morrison (Fritz Lipmann Institute)
|
| Treatment protocol |
SC4 cells were passaged into 6-well plates at a concentration of 5.0 x 10^4 cells/well and were grown for 24 hours before treatment. siRNAs were transfected in OptiMem(Life Technologies) using Lipofectamine 2000 reagent(Life Technologies) at a final concentration of 40 nL of siRNA . Transfected cells were grown for 48 hours before RNA extraction.
|
| Growth protocol |
SC4 cells were maintained in DMEM media with 10% FBS at 37 degrees C, and 5% CO2. Standard cell culture techniques were employed.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Media was aspirated and cells were washed twice with sterile PBS. 0.5 ml of Trizol reagent (Life Technologies) was added to each well. Cell lysates were collected and frozen immediately and stored at -80 degrees C overnight.
For RNA-Seq analysis, 5-10 μg of total RNA was used to isolate poly(A) RNA using the micropoly(A)purist kit (Ambion). The whole transcriptome library kit (Life Technologies) was used to prepare paired-end sequencing libraries. Briefly, 100 ng of poly(A)+ RNA was enzymatically fragmented to an average size of 150 nt, ligated to directional adapters and reverse-transcribed, and the cDNA was size-selected on denaturing 6% TBE-Urea polyacrylamide gels, amplified for 15 cycles with barcoded primers and purified using AmpPure XP beads. The resulting libraries were quantified using Bioanalyzer (Agilent) and Taqman (Life Technologies) assays. Barcoded libraries from each sample were combined at equimolar amounts and the mixture was used at a final concentration of 0.4 pM in emulsion PCR to link the libraries to the sequencing beads.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
AB 5500 Genetic Analyzer |
| |
|
| Description |
Mouse cells isolated from Nf2-null schwannoma
|
| Data processing |
Adaptor trimming was performed by 'flexbar 2.41 beta' for csfasta files and using an internal perl script for qual files
Quality trimming was performed using an internal perl script
Reads were aligned to mm9 reference genome using 'Tophat 2.0.9'
Raw read counts generated using HTSeq 0.6.1
Normalized counts and a list of differentially expressed genes generated using DESeq 1.14.0
Genome_build: mm9
Supplementary_files_format_and_content: Processed data file is a text file and contains normalized counts obtained from DESeq 1.14.0
|
| |
|
| Submission date |
Sep 18, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Joseph Kissil |
| E-mail(s) |
jkissil@scripps.edu
|
| Phone |
(561) 228-2170
|
| Organization name |
The Scripps Research Institute
|
| Department |
Cancer Biology
|
| Street address |
130 Scripps Way
|
| City |
Jupiter |
| State/province |
Florida |
| ZIP/Postal code |
33458 |
| Country |
USA |
| |
|
| Platform ID |
GPL16790 |
| Series (1) |
| GSE61528 |
YAP mediates tumorigenesis in neurofibromatosis type 2 through a COX2-EGFR signaling axis |
|
| Relations |
| BioSample |
SAMN03073524 |
| SRA |
SRX703749 |
