|
| Status |
Public on Mar 16, 2017 |
| Title |
SAM822639 Crushed.Cre- |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
RNA from whole retinas 3 days after optic nerve crush
|
| Organism |
Mus musculus |
| Characteristics |
tissue: retina
samid: SAM822639
treatment: Optic nerve crushed; Cre-
|
| Treatment protocol |
To investigate the role of DLK after optic nerve crush, we generated mice with a tamoxifen-inducible Cre recombinase-estrogen receptor (Cre-ERT) transgene driven by the chicken beta-actin-CMV hybrid (CAG) promoter, resulting in high levels of Cre-ERT in many tissues, including retina. CAG Cre-ERT:DLKlox/lox (referred to as DLKlox:Crepos) mice survive to adulthood with no evidence of the developmental abnormalities seen in DLK-null animals (8, 18). Dosing of DLKlox:Crepos mice at 10-12 wk of age with tamoxifen (Fig. S2A) results in elimination of the majority of DLK expression in retina (Fig. 1 C and L), with the small amount of remaining DLK protein varying slightly from animal to animal (Fig. 1C and Fig. S2B). No differences in health or behavior were observed between tamoxifen-treated DLKlox:Crepos mice and their tamoxifen-treated DLKlox:Creneg control littermates.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted from whole retina as described in Watkins et al. PNAS. (2013). RNA sample quantity and quality were determined using an ND-1000 spectrophotometer (Thermo Scientific) and Bioanalyzer 2100 (Agilent Technologies), respectively.
|
| Label |
Cy5
|
| Label protocol |
The method for preparation of Cy-dye labeled cRNA and array hybridization was provided by Agilent. Briefly, 1 μg total RNA was converted to double-stranded cDNA and then to Cy5-labeled cRNA using Quick Amp Labeling Kit (Agilent). The labeled cRNA was purified using RNeasy mini kit (Qiagen). cRNA yield and Cy5 incorporation was determined using ND-1000 spectrophotometer.
|
| |
|
| Channel 2 |
| Source name |
universal mouse reference (Stratagene)
|
| Organism |
Mus musculus |
| Characteristics |
samid: NA
treatment: NA
|
| Extracted molecule |
total RNA |
| Extraction protocol |
By vendor
|
| Label |
Cy3
|
| Label protocol |
Method for Cy-dye labeled cRNA preparation was provided by Agilent.
|
| |
|
| |
| Hybridization protocol |
750 ng of labeled cRNA was fragmented and hybridized to Agilent’s Whole Mouse Genome 4x44Kv2 arrays as described in manufacturer’s hybridization kit, against an equal amount of Cy3-labeled universal mouse reference (Stratagene).
|
| Scan protocol |
Following hybridization, microarrays were washed, dried, and scanned on Agilent’s G2505C scanner. Agilent’s Feature Extraction 11.5 software was used to analyze acquired array images.
|
| Description |
SAM822639 Crushed.dlkPlus
|
| Data processing |
Data were analyzed using the limma package from Bioconductor. ControlType weights were set to 0, spots with background signal more than 50 above the foreground signal were set to the median background intensity, background correction was performed with limma::backgroundCorrect() using the "normexp" method and an offset of 50, limma::normalizeWithinArrays() with the "loess" method, and finally limma::normalizeBetweenArrays() with the "Aquantile" method. This gives the "test" and "reference" normalized probe intensities. Finally, for each probe an "expression ratio" was calculated, the ratio of the normalized signals in the test and reference channels.
|
| |
|
| Submission date |
Mar 09, 2017 |
| Last update date |
Mar 17, 2017 |
| Contact name |
Kevin Huang |
| E-mail(s) |
huang.kevin@gene.com
|
| Organization name |
Genentech
|
| Department |
Bioinformatics
|
| Street address |
1 DNA Way
|
| City |
South San Francisco |
| State/province |
California |
| ZIP/Postal code |
94080 |
| Country |
USA |
| |
|
| Platform ID |
GPL11202 |
| Series (1) |
| GSE96053 |
Dlk response to optic nerve injury |
|
