population: VA arsenic exposure: + hours exposed to fresh water: 0 tissue: gill slide number: 4
Growth protocol
Fish were pre-acclimated to common garden conditions, housed, maintained, and exposed to arsenic as described in Shaw, J. R., Hampton, T. H., et al. (2014). Natural selection canalizes expression variation of environmentally induced plasticity-enabling genes. Molecular Biology and Evolution, 31(11), 3002–3015. 72 killifish were acclimated to seawater for two weeks. Of these, 36 fish (12 from each of the three populations) were exposed to freshwater for 0 h, 1 h, or 24 h, and an additional 36 fish were exposed to the same time course in the presence of 100 micrograms per liter of arsenic.
Extracted molecule
total RNA
Extraction protocol
RNAeasy kits were used for RNA extraction (Qiagen, Valencia, CA). Tissue samples were removed from RNA-later and rinsed before being homogenized using a Tissue-Tearor (Biospec Products, Barlesville, OK). The homogenized tissue was then processed using RNeasy kits with DNase treatment (Qiagen, Valencia, CA) to extract RNA. RNA quality and concentration was determined by analysis with an Agilent 2100 bioanalyzer at the Harvard-Partners Center for Genetics and Genomics (Harvard Medical School, Cambridge, MA USA).
Label
cy3
Label protocol
RNA was amplified using MessageAmp II kits (Ambion) and aRNA labeled using a single channel design with Cy3 dye using NimbleGen Labeling Kit (Roche NimbleGen)
Hybridization protocol
Labelled samples were randomly hybridized using the Hybridization Kit (Roche NimbleGen) to a custom NimbleGen array whose design and annotation has been previously described (Shaw et al. 2014). Briefly, this platform interrogates 135,000 probes that map to 34,713 gene models and 16,104 unique Fundulus heteroclitus genes from a high-quality killifish reference genome (Reid et al., 2017)
Scan protocol
Scanning was performed by NimbleGen Systems Inc., Madison, WI USA, following their standard operating protocol. Raw data were extracted using NimbleScan v2.4 software (Roche NimbleGen)
Description
A8_2 This sample is of gill tissue from saltwater acclimated Kings Creek killifish not exposed to freshwater that were exposed to 100ug/l arsenic, replicate 4
Data processing
The raw data (.pair file) was subjected to RMA (Robust Multi-Array Analysis; Irizarry et al. Biostatistics 4(2):249), quantile normalization (Bolstad et al. Bioinformatics 19(2):185), and background correction as implemented in the NimbleScan software package, version 2.4.27 (Roche NimbleGen, Inc.).
