|
| Status |
Public on Sep 14, 2020 |
| Title |
C.elegans_WT_adult_worm_rep2 |
| Sample type |
RNA |
| |
|
| Source name |
N2_adult_worm_replicate2
|
| Organism |
Caenorhabditis elegans |
| Characteristics |
developmental stage: young_adult
genotype: wild-type
|
| Growth protocol |
Embryos were isolated from gravid adults by hypochlorite disruption (0.5N NaOH and 1% NaOCl) followed by M9 washes. After overnight incubations in M9 without food, synchronized L1 were then seeded on a 5.5cm NGM plate (~150 L1 /plate). The synchronized young adult (52 hrs post L1 feeding) were harvested and washed by M9 buffer and pelleted. Embryos were isolated by hypochlorite disruption from young gravid adult (52 hrs after L1), washed with M9, and pelleted
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Synchronized young adult or embryos derived from ~40,000 young adults were resuspended in 500 μl TRIzol reagent followed by flash freezing in liquid nitrogen. Samples were thawed at 42°C and frozen in liquid nitrogen and the cycle was repeated 6-7 times. 100 μl chloroform was mixed with sample and spun at 12,000g for 15 minutes at 4°C. The corresponding aqueous layer was harvested and mixed with equal volume of 70% ethanol. RNeasy Mini kits (Qiagen) were used to obtain pure RNA for microarray analysis.
|
| Label |
Cy3
|
| Label protocol |
A 0.2 μg of total RNA was subjected to Cy3 labeling by in vitro transcription with use of Low Input Quick-Amp Labeling kit (Agilent Technologies, USA).
|
| |
|
| Hybridization protocol |
1.65 μg of Cy3-labled cRNA was incubated at 60°C for 30 minutes, followed by hybridization to the Agilent C. elegans V2 4*44K Microarray chip (Agilent Technologies, USA) at 65°C for 17 hrs. 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 |
The microarray chip was scanned with the microarray scanner (Agilent Technologies, USA) at 535 nm for Cy3
|
| Description |
stage:52 hours after L1
|
| Data processing |
The array image was read by Feature Extraction software version 10.7.1.1, and data was subjected to further analysis by GeneSpring software (Agilent) by following parameter:Threshold: 1.0; Logbase: 2; Technology: Agilent.SingleColor.20186; Normalization: Shift to 75 percentile
Average value of N2 sample was used as the control
|
| |
|
| Submission date |
Sep 11, 2020 |
| Last update date |
Sep 15, 2020 |
| Contact name |
Leng-Jie Huang |
| E-mail(s) |
kkk12332141@gate.sinica.edu.tw
|
| Organization name |
Academia Sinica
|
| Department |
Institute of Plant and Microbial Biology
|
| Street address |
128 Sec. 2, Academia Rd, Nankang
|
| City |
Taipei |
| ZIP/Postal code |
11529 |
| Country |
Taiwan |
| |
|
| Platform ID |
GPL11346 |
| Series (1) |
| GSE157800 |
Loss of the seipin gene perturbs eggshell formation in Caenorhabditis elegans |
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