|
| Status |
Public on Aug 08, 2016 |
| Title |
Cypermethrin_copepodid_rep2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Copepodid lice (Atlantic Canada)
|
| Organism |
Lepeophtheirus salmonis |
| Characteristics |
cypermethrin concentaion (ppb): 1
|
| Treatment protocol |
Stock cyeprmethrin was prepared to 1 mg/L using fresh sea water. Stock solution was further diluted to 10 ug/L for a working solution. A 10-fold dilution of cypermethrin working solution was then used for all exposures. Final cypermethrin concentration was 1.0 ppb (ug/L) with n = 6 pools of 500 copepodids. Exposure time was 24 hours before collection using 80um mesh filters and scraping copepodids into a tube (~500 copepodids/tube) and kept at -80 degrees Celcius until RNA extraction (12 individual extractions).
|
| Growth protocol |
Sea lice (L. salmonis) were collected from BMA2a in the Bay of Fundy, New Brunswick (NB) Canada in July 2014. Gravid females were returned to the Huntsman Marine Science Center, NB in chilled coolers for egg string removal and culture. Egg strings were placed in a flow-through hatch system and allowed to develop to the infective copepodid stage. At this time, lice were transported to the Atlantic Veterinary College, UPEI for in vitro bioassay procedures.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from frozen tissue using TRIzol reagent® (Life Technologies), on-column DNase digestion (QIAGEN) followed by RNEasy column purification as per manufacturers’ instructions (QIAGEN). Purified total RNA was tested by agarose gel and automated electrophoresis (Experion; Bio-Rad) and spec.
|
| Label |
Cy5-CTP
|
| Label protocol |
Labeled cRNA was generated from 825ng total RNA using Low-Input Quick Amp kits (Agilent; v6.5). A Cy3-cRNA reference pool to hybridize alongside samples was created for each of the above experiments, ensuring each experimental condition was included in the pool.
|
| |
|
| Channel 2 |
| Source name |
all condition reference pool
|
| Organism |
Lepeophtheirus salmonis |
| Characteristics |
sample type: reference pool
|
| Treatment protocol |
Stock cyeprmethrin was prepared to 1 mg/L using fresh sea water. Stock solution was further diluted to 10 ug/L for a working solution. A 10-fold dilution of cypermethrin working solution was then used for all exposures. Final cypermethrin concentration was 1.0 ppb (ug/L) with n = 6 pools of 500 copepodids. Exposure time was 24 hours before collection using 80um mesh filters and scraping copepodids into a tube (~500 copepodids/tube) and kept at -80 degrees Celcius until RNA extraction (12 individual extractions).
|
| Growth protocol |
Sea lice (L. salmonis) were collected from BMA2a in the Bay of Fundy, New Brunswick (NB) Canada in July 2014. Gravid females were returned to the Huntsman Marine Science Center, NB in chilled coolers for egg string removal and culture. Egg strings were placed in a flow-through hatch system and allowed to develop to the infective copepodid stage. At this time, lice were transported to the Atlantic Veterinary College, UPEI for in vitro bioassay procedures.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from frozen tissue using TRIzol reagent® (Life Technologies), on-column DNase digestion (QIAGEN) followed by RNEasy column purification as per manufacturers’ instructions (QIAGEN). Purified total RNA was tested by agarose gel and automated electrophoresis (Experion; Bio-Rad) and spec.
|
| Label |
Cy3-CTP
|
| Label protocol |
Labeled cRNA was generated from 825ng total RNA using Low-Input Quick Amp kits (Agilent; v6.5). A Cy3-cRNA reference pool to hybridize alongside samples was created for each of the above experiments, ensuring each experimental condition was included in the pool.
|
| |
|
| |
| Hybridization protocol |
Samples were hybridized to oligonucleotide microarrays as per manufacturers' instructions for Low-Input Quick Amp kits (Agilent; v6.5). Arrays were designed using previously annotated ESTs from both Pacific and Atlantic L. salmonis (Yasuike et al. 2012; eArray design ID 024389; Agilent).
|
| Scan protocol |
Scanned at 5 micrometer resolution on a ScanArray Express (Perkin Elmer) and quantified on Imagene (v8.1; BioDiscovery).
|
| Data processing |
For each probe on the microarray, the background median was subtracted from the foreground median. Data analysis was performed in GeneSpring GX11 (Agilent). Each array was normalized using an intensity-dependent Lowess normalization. All entities on the array are presented in the attached matrix file here, however, for the analysis, quality control filters for each of the three experiments retained probes that pass the following criteria in at least 65% of the samples in any one condition: raw signal ≥ 500 in both channels and no poor quality flags.
|
| |
|
| Submission date |
Jan 05, 2016 |
| Last update date |
Aug 09, 2016 |
| Contact name |
Ben F Koop |
| E-mail(s) |
bkoop@uvic.ca
|
| Phone |
(250) 472-4067
|
| Organization name |
The University of Victoria
|
| Department |
Biology
|
| Lab |
Centre for Biomedical Research
|
| Street address |
PO Box 3020 STN CSC
|
| City |
Victoria |
| State/province |
BC |
| ZIP/Postal code |
V8W 3N5 |
| Country |
Canada |
| |
|
| Platform ID |
GPL15566 |
| Series (1) |
| GSE76555 |
Transcriptomic responses of copepodid sea lice (Lepeophtheirus salmonis) exposed to cypermethrin (Betamax®) |
|
