|
| Status |
Public on Mar 02, 2012 |
| Title |
Translatome NIH3T3 cells control |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
NIH3T3 cells
|
| Organism |
Mus musculus |
| Characteristics |
cell line: NIH3T3 cells
rna fraction: polysomal RNA (P)
|
| Treatment protocol |
Where indicated, the cells were treated with 10microM thapsigargin (# T9033, Sigma) for 3h.
|
| Growth protocol |
Four subconfluent p100 plates of NIH3T3 cells were grown in DMEM +10% bovine serum (#16170-078, Gibco).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The cells were lysed and polysome bound RNA was separated by centrifugation in a 10-40% sucrose gradient. Fractions were extracted with phenol, ethanol precipitated and pooled in two fractions: free+monosome (FM) fraction, and polysome bound (P) fraction.
|
| Label |
Cy3
|
| Label protocol |
For labeling, we used equal volumes of FM and P fractions so that the total amount of RNA for each sample was adjusted to 300 ng. Polysome bound RNA was labeled with Cy3 and RNA of FM fraction was labeled with Cy5 using the low imput Quick Amp Labelling Kit, Two-Color (Agilent p/n 5190-2306) according to the manufacturer’s instructions.
|
| |
|
| Channel 2 |
| Source name |
NIH3T3 cells
|
| Organism |
Mus musculus |
| Characteristics |
cell line: NIH3T3 cells
rna fraction: non-polysomal RNA (FM)
|
| Treatment protocol |
Where indicated, the cells were treated with 10microM thapsigargin (# T9033, Sigma) for 3h.
|
| Growth protocol |
Four subconfluent p100 plates of NIH3T3 cells were grown in DMEM +10% bovine serum (#16170-078, Gibco).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The cells were lysed and polysome bound RNA was separated by centrifugation in a 10-40% sucrose gradient. Fractions were extracted with phenol, ethanol precipitated and pooled in two fractions: free+monosome (FM) fraction, and polysome bound (P) fraction.
|
| Label |
Cy5
|
| Label protocol |
For labeling, we used equal volumes of FM and P fractions so that the total amount of RNA for each sample was adjusted to 300 ng. Polysome bound RNA was labeled with Cy3 and RNA of FM fraction was labeled with Cy5 using the low imput Quick Amp Labelling Kit, Two-Color (Agilent p/n 5190-2306) according to the manufacturer’s instructions.
|
| |
|
| |
| Hybridization protocol |
Equal volumes of labeled cRNA product were co-hybridized with Mouse Gene Expression Microarray (Agilent p/n G2519F-014868).
|
| Scan protocol |
Scanned on Agilent Technologies Scanner G2505B US45102947
|
| Description |
biological replicate 1 of 1. Translatome of control NIH3T3 cells
|
| Data processing |
Data extracted using Agilent Feature Extraction Software 10.7.1 using the grid template 014868_D_F_20100123 following the Agilent protocol GE2_107_Sep09 and the QC Metric Set GE2_QCMT_Sep09. Half background substration was carried in Babelomics 4.0 suite. Probe replicates were averaged. For processed data, only coding mRNAs are shown.
Translation efficiencies are in log2 ratio (Cy5/Cy3) for rawdata, and log2 ratio (Cy3/Cy5) for processed data.
|
| |
|
| Submission date |
Mar 01, 2012 |
| Last update date |
Mar 06, 2012 |
| Contact name |
Ivan Ventoso |
| E-mail(s) |
iventoso@cbm.uam.es
|
| Phone |
34-911964809
|
| Fax |
34-911964420
|
| Organization name |
Centro de Biología Molecular Severo Ochoa
|
| Department |
Virology and Microbiology
|
| Lab |
126
|
| Street address |
Nicolas cabrera 1. Universidad Autonoma de Madrid
|
| City |
Madrid |
| State/province |
Madrid |
| ZIP/Postal code |
28049 |
| Country |
Spain |
| |
|
| Platform ID |
GPL4134 |
| Series (1) |
| GSE36206 |
Translatome of NIH3T3 cells: control vs. stress treatment |
|
