|
| Status |
Public on Jun 01, 2016 |
| Title |
Female_0.01_rep3 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Female_pre-adult lice_0.01
|
| Organism |
Lepeophtheirus salmonis |
| Characteristics |
Sex: Female
age: pre-adult lice
population: Pacific Canada
microsporidian identification (facilispora margolisi): Positive
emb concentration (ppb): 0.01
|
| Treatment protocol |
Stock emamectin benzoate (EMB; PESTANAL®, Sigma-Aldrich) was prepared to 100 mg/L in methanol, then diluted to working concentrations in seawater. EMB dilutions were prepared to final concentrations of 0, 0.01, and 0.01 parts per billion (n = 6-8 individuals for each sex/dose combination). Exposure time was 24 hours before collection and flash freezing.
|
| Growth protocol |
Pacific: L. salmonis egg strings were obtained from adult female lice infecting Atlantic salmon in a salmon farm near Broughton Archipegalo, British Columbia (BC) in 2010. Eggs were hatched in a static sea water hatch system, grown to copepodids, and then grown on Atlantic salmon in house at Pacific Biological Station, Nanaimo, BC, Canada. Pre-adult lice were removed from Atlantic salmon sedated with Metomidate hydrochloride (Aquacalm, Syndel Laboratories Ltd.) A total of 40 pre-adult I and II stage L. salmonis individuals were randomly distributed into beakers containing 500 ml filtered seawater (10C; 30 parts per thousand salinity; one air stone per beaker).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Lice were flash frozen individually and kept at -80 degrees Celcius until RNA extraction (n = 40 individuals). 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 pool
|
| Organism |
Lepeophtheirus salmonis |
| Characteristics |
sample type: pooled reference
|
| Treatment protocol |
Stock emamectin benzoate (EMB; PESTANAL®, Sigma-Aldrich) was prepared to 100 mg/L in methanol, then diluted to working concentrations in seawater. EMB dilutions were prepared to final concentrations of 0, 0.01, and 0.01 parts per billion (n = 6-8 individuals for each sex/dose combination). Exposure time was 24 hours before collection and flash freezing.
|
| Growth protocol |
Pacific: L. salmonis egg strings were obtained from adult female lice infecting Atlantic salmon in a salmon farm near Broughton Archipegalo, British Columbia (BC) in 2010. Eggs were hatched in a static sea water hatch system, grown to copepodids, and then grown on Atlantic salmon in house at Pacific Biological Station, Nanaimo, BC, Canada. Pre-adult lice were removed from Atlantic salmon sedated with Metomidate hydrochloride (Aquacalm, Syndel Laboratories Ltd.) A total of 40 pre-adult I and II stage L. salmonis individuals were randomly distributed into beakers containing 500 ml filtered seawater (10C; 30 parts per thousand salinity; one air stone per beaker).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Lice were flash frozen individually and kept at -80 degrees Celcius until RNA extraction (n = 40 individuals). 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).
|
| Description |
raw data files: 10079_cy5_70txt; 10079_cy3_75txt
10079_Block2
|
| 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 |
Oct 05, 2015 |
| Last update date |
Jun 01, 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) |
| GSE73734 |
Sex-Biased Gene Expression and Sequence Conservation in Pacific and Atlantic Salmon Lice (Lepeophtheirus salmonis) |
|
