|
| Status |
Public on Jul 27, 2016 |
| Title |
Knockout_rep4 |
| Sample type |
RNA |
| |
|
| Source name |
midbrain, adult (8-9 weeks), agouti homozygous mutant, MSM background
|
| Organism |
Mus musculus molossinus |
| Characteristics |
tissue: midbrain
developmental stage: adult (8-9 weeks)
gender: male
strain: MSM/Ms
agouti (a) allele: homzygous mutant
|
| Treatment protocol |
The midbrain tissues were manually isolated from 8-9 weeks old mice under dissecting microscope.
|
| Growth protocol |
MSM/Ms mice were derived from the colony maintained at RIKEN BioResource Center. The mutant mice for agouti (a) allele were generated by using CRISPR/Cas9 system. They were housed under controlled lighting conditions (daily light period 07:00 to 21:00).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was purified from isolated midbrain tissues with Trizol (Life technologies). The concentration of RNA was quantified using a NanoDrop-1000 spectrophotometer.
|
| Label |
Cy3
|
| Label protocol |
cRNA was amplified from 0.1 ug of RNA and labeled with Cy3 using Low-Input QuickAmp Labeling Kit (Agilent Technologies) according to the manufacturer's instructions, followed by RNAeasy MinElute kit (QIAGEN).. Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
|
| |
|
| Hybridization protocol |
0.6 ug of Cy3-labelled cRNA was fragmented at 60°C for 30 minutes in a reaction volume of 25 ul following the manufacturers instructions. On completion of the fragmentation reaction, 25 ul of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to SurePrint G3 Mouse Gene Expression v2 8x60K Microarray (G4852B) at 65°C for 17 h in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent), then dried immediately.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505C) using one color scan setting for 8x60k array slides (Scan Area 61x21.6 mm, Scan resolution 3um, Dye channel is set to Green).
|
| Data processing |
The scanned images of microarray slides were processed using Feature Extraction software version 10.5.1.1 (Agilent Technologies). All raw data were loaded into Gene Spring GX 13.1.1 (Agilent Technologies) and signal intensities were normalized by shift to 75 percentile. Baseline was set to the median of all samples. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
|
| |
|
| Submission date |
Jul 26, 2016 |
| Last update date |
Jul 27, 2016 |
| Contact name |
Shogo Matoba |
| E-mail(s) |
shogomatoba@gmail.com
|
| Organization name |
RIKEN BioResource Research Center
|
| Street address |
3-1-1, Koyadai
|
| City |
Tsukuba |
| State/province |
Ibaraki |
| ZIP/Postal code |
305-0074 |
| Country |
Japan |
| |
|
| Platform ID |
GPL21163 |
| Series (1) |
| GSE84840 |
CRISPR/Cas9-mediated genome editing in wild-derived mice; Generation of tamed wild-derived strains by mutation of the a (nonagouti) gene |
|
