|
| Status |
Public on Feb 21, 2023 |
| Title |
βB2-crystallin W151C knock-in Replicate 2 |
| Sample type |
RNA |
| |
|
| Source name |
βB2-crystallin W151C knock-in mouse lens
|
| Organism |
Mus musculus |
| Characteristics |
age: 3 months old
tissue: lens
genotype: BetaB2-crystallin W151C knock-in
|
| Treatment protocol |
BetaB2-W151C knock-in mice expressing an G to C point mutation in codon 151 of the mouse βB2-crystallin gene CRYBB2, which results in the substitution of Tryptophan 151 with Cysteine (W151C), were generated using CRISPR/Cas9 system by Shanghai Biomodel Organisms Science & Technology Development Co., Ltd. (Shanghai, China)
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was prepared using TRIzol® Reagent (Invitrogen life technologies) following the manufacturer's recommendations. RNA was cleaned up using RNasey Mini Kit(Qiagen p/n 74104) and was quantified using a NanoDrop-1000 spectrophotometer.
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 1.0 ug RNA using Quick Amp Labeling Kit, One-Color (Agilent p/n 5190-0442) according to the manufacturer's instructions, followed by RNAeasy column purification (Qiagen p/n 74104). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
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| |
|
| Hybridization protocol |
1.5 ug of Cy3-labelled cRNA (specific activity >10.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 250 ml containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 250 ml of 2x Agilent Gene Expression hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Whole Human Genome Oligo Microarrays (G4112A) for 17 hours at 65°C 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 by brief centrifugation.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2565BA) using one color scan setting for 1x44k array slides (Scan Area 61x21.6 mm, Scan resolution 5um, Dye channel is set to Green and Green PMT is set to 100%).
|
| Description |
Gene expression in βB2-crystallin W151C mutant lens at 3 months old
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 9.1 (Agilent) using default parameters (protocol GE1-v1_91 and Grid: 012391_D_20060331) to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
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| |
|
| Submission date |
Feb 17, 2023 |
| Last update date |
Feb 22, 2023 |
| Contact name |
Wei Xiao |
| E-mail(s) |
xiaow27@mail.sysu.edu.cn
|
| Phone |
15989187341
|
| Organization name |
Zhongshan Ophthalmic Center
|
| Lab |
State Key Laboratory of Ophthalmology
|
| Street address |
S 54 Xianlie Rd, Yuexiu District
|
| City |
Guangzhou |
| State/province |
Guangdong |
| ZIP/Postal code |
510060 |
| Country |
China |
| |
|
| Platform ID |
GPL11202 |
| Series (1) |
| GSE225548 |
Murine lenses:βB2-crystallin W151C knock-in mice vs.wild type control mice |
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