normalization data transformation protocol
Any spots with an intensity of less than 300 (the background level on the arrays) were removed from the analyses, as were spots present on less than 20 out of 24 arrays. Expression data were log-transformed. Please note identifiers in this file map to the Comment[PROBE_ID] column of the array design file.
array scanning protocol
Arrays were scanned using Axon GenePix 4000B with the software NimbleScan (version=2.4.27). (Parameters: Scanning hardware = Axon- GenePix4000B, Scanning software = Scanning software)
nucleic acid labeling protocol
15 ug of aRNA were dyed with Cy3 or Cy5 (GE Health Care #RPN5661) and subsequently purified according to the Ambion Kit instructions. 1.5 ug of a Cy3 labeled sample were combined with 1.5 ug of a Cy5 labeled sample and fragmented using RNA Fragmentation Reagents (Ambion AM8740) according to the manufacturers instructions. (Parameters: Amount of nucleic acid labeled = 15, Amplification = OTHER: Ambion (Life Technologies) Amino Allyl MessageAmp II aRNA Amplification Kit (AM1753), Mass unit = Micro gram)
Newly mated queens of S. invicta were set up in pairs (pleometrosis) based on having similar weights (range ±0.2 mg) and paint-marked with different colors, or individually (haplometrosis) and paint-marked as well. All the queens were provided with a nesting chamber consisting of a glass tube half-filled with water, which was covered by a cotton ball and a layer of dental plaster: this keeps the chamber moist but avoids an excess of water which is deleterious for the young brood. Tubes were sealed with a loose cap to provide air flow. Specimens were reared in the dark at 28C, 70% relative humidity under claustral conditions (no food and no water) for 1 month. After the eclosion of the first batch of workers (minims), incipient colonies were provided with water, sugar water and frozen crickets. Glass tubes were set open in pencil boxes coated with Fluon to prevent escape. (Parameters: time unit = seconds, temperature unit = C)
679 newly mated queens of S. invicta were split into two groups: 308 queens were set up in pairs (pleometrosis) based on having similar weights (range ±0.2 mg) and paint-marked with different colors, while 371 queens were set up individually (haplometrosis) and paint-marked as well. Pleometrotic queens included queens that survive the competition (winners), usually located at the top of the brood pile within the nest chamber and generally tended by workers; and queens that will be executed (losers), normally seen outside the nest chamber, hiding from workers in order to avoid being attacked.
nucleic acid extraction protocol
Total RNA from individual fire ant queens was extracted using the RNeasy Plus kit (Qiagen) combined with a RNase-Free DNase step (Qiagen) to remove any possible contamination by genomic DNA. RNA concentration and purity were assessed using NanoDrop and Qubit and RNA quality was assessed using RNA Nano Chips on the Agilent Bioanalyzer. 1ug of each sample was amplified using the Ambion (Life Technologies) Amino Allyl MessageAmp II aRNA Amplification Kit (AM1753). (Parameters: Extracted product = total_RNA, Amplification = OTHER: Ambion (Life Technologies) Amino Allyl MessageAmp II aRNA Amplification Kit (AM1753))