flies were reared on cornmeal/yeast medium and maintained at 20°C with a 12:12-h light/dark cycle.
Extracted molecule
total RNA
Extraction protocol
flies were instantly frozen in liquid nitrogen and stored in TRIZOL (Invitrogen) at -80°C. RNA was isolated using TRIZOL from at least 40 individuals. RNA quantity and quality were separately measured on a NanoDrop spectrophotometer (Thermo Scientific) and a Bioanalyzer 2100 (Agilent Technologies). High-quality samples (260/280>1.85; 260/230>1.7) from all lines of each species were pooled in equal amounts and further purified using the RNeasy kit (Qiagen).
Label
Cy3
Label protocol
Fluorescent cRNA was synthesized from 200 ng of total RNA using Agilent’s low-RNA input fluorescent linear amplification kit following manufacturer's protocols (Agilent Technologies). Labelled RNAs were cleaned with RNeasy columns (Qiagen), and cRNA yields were quantified on a NanoDrop spectrophotometer. Two separate labeling reactions per sample were pooled and hybridized to the arrays following Agilent’s protocols.
Hybridization protocol
Agilent’s protocols
Scan protocol
Agilent’s protocols
Description
Global gene expression of 7-day-old virgin adults
Data processing
Performed in HDBStat: data is normalized by flagging out bad spots, filtering unexpressed genes (average raw intensity across all the samples <50), negative and positive Agilent controls, and then normalized with Chip Mean and log2 transformation.
Evolution of sex-dependent gene expression in three recently diverged species of Drosophila
Data table header descriptions
ID_REF
VALUE
normalized data after flagging out bad spots, filtering unexpressed genes (average raw intensity across all the samples <50), negative and positive Agilent controls, and then normalized with Chip Mean and log2 transformation.
