|
| Status |
Public on Jul 14, 2016 |
| Title |
control-shRNA rep2 |
| Sample type |
RNA |
| |
|
| Source name |
E14-control
|
| Organism |
Mus musculus |
| Characteristics |
cell type: E14 embryonic stem cell from ATCC (CRL-1821)
transfected with: Control non-specific shRNA
genotype/variation: wild type
time point: passage 3 after each transfection
|
| Treatment protocol |
Control non-specific shRNA and Tip5 shRNA coned into pSuper vector were electroporated into E14 cells using Amaxa Nucleofector. To increase the efficiency of Tip5 knockdown, combination of shRNA constructs in different selection markers were used. Seventy-two hours later, transfected cells were further selected for 6 days with appropriate antibiotics.
|
| Growth protocol |
E14 cells were maintained in ES medium (knock-out DMEM, 20% fetal bovine serum, 55 µM mercaptoethanol, 1 mM sodium pyruvate, 2 mM glutamine, 0.1 mM nonessential amino acids, and 1000 U/ml mouse LIF), on tissue culture plates coated with 0.1% gelatin, in 6% CO2 at 37°C.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was purified using TRI reagent (Sigma) and further cleaned using the RNeasy kit (Qiagen). The quality of total RNA was confirmed on a Bioanalyzer 2100 (Agilent Technologies, Inc.).
|
| Label |
biotin
|
| Label protocol |
Biotinylated cRNA were prepared with the Ambion MessageAmp kit for Illumina arrays
|
| |
|
| Hybridization protocol |
Standard Illumina hybridization protocol
|
| Scan protocol |
Standard Illumina scanning protocol
|
| Description |
SAMPLE 2
|
| Data processing |
The data were normalized using average normalization and subtracting background parameters with GenomeStudio V2011.1. To remove probes for targets that were likely not significant, we consider the intensity value as “1” if signal intensity is less than 1.
Average fold was calculated by the each triplicate ratio of Tip5-shRNA vs control-shRNA (average_fold_data.txt).
|
| |
|
| Submission date |
Jul 15, 2015 |
| Last update date |
Jul 14, 2016 |
| Contact name |
Hunter Richards |
| E-mail(s) |
hrichards@ucsf.edu
|
| Organization name |
UCSF
|
| Department |
Orofacial Sciences
|
| Lab |
Kohwi-Shigematsu
|
| Street address |
513 Parnassus Ave., HSW 863
|
| City |
San Francisco |
| State/province |
CA |
| ZIP/Postal code |
94103 |
| Country |
USA |
| |
|
| Platform ID |
GPL6885 |
| Series (2) |
| GSE70969 |
Tip5/Baz2a Regulates Chromatin Architecture and Gene Expression to Maintain Self-renewal and Pluripotency of Embryonic Stem Cells [microarray] |
| GSE70975 |
Tip5/Baz2a Regulates Chromatin Architecture and Gene Expression to Maintain Self-renewal and Pluripotency of Embryonic Stem Cells. |
|
