Transcriptional effects of diacetyl exposure

Flies were exposed to diacetyl (B85307, Sigma-Aldrich, St. Louis, MO) by placing them in vials in a cylindrical closed container (112 mm diameter × 151 mm height) along with an odor-containing glass vial. The odorant was dissolved in 5 mL paraffin oil at 1% dilution. For a given exposure protocol, two groups of flies were prepared: those exposed to 1% diacetyl headspace and those exposed to paraffin oil headspace alone (control flies). Adult male flies aged 1 d were transferred to fly vials containing fresh medium and put into the container with the odor vial. At the end of the fifth day of exposure, flies were collected, and their antennae were dissected for RNA extraction. All treatments and experiments were performed at room temperature. For the recovery experiment, flies were transferred to a container with a glass vial of paraffin oil after 5 days of diacetyl exposure. At the end of the fifth day of recovery, flies were collected, and their antennae were dissected for total RNA extraction. The second and third antennal segments from 40–60 male flies after treatment were carefully hand-dissected from the head and collected in 1.5 ml microfuge tubes kept cold in liquid nitrogen. Antennae were mechanically crushed with disposable RNAse-free plastic pestles, and total RNA was isolated using a Trizol-based protocol. cDNA libraries were prepared from total RNA using the Illumina TruSeq RNA Sample Preparation Kit (v2) and 50 bps single- and paired-end sequencing was done using the HighSeq2000. Two biological replicates were sequenced for each condition, with an average of 27 million reads / replicate, and with an average of 84% mapped.
Two-month old C57BL/6 male mice (2–3 for each condition in a single cage) were continually exposed to air flowing over headspace of paraffin oil (solvent control), 0.1%, or 1% diacetyl over a period of 5 days, then euthanized for collecting the lung and brain tissues and processing for mRNA isolation. All protocols for animal use and euthanasia were approved by the Institutional Animal Care and Use Committee (#20150028) and were in accordance with the provisions established by the Animal Welfare Act and the Public Health Services (PHS) Policy. In the transcriptome analysis, two replicates were performed for each condition, with an average of 123,687,411 reads / replicate, with an average of 88% mapped. Multiplexed libraries were made from total RNA input using the Illumina TruSeq RNA sample preparation kit (v2) and 50 bps single-end sequencing was done using the NextSeq500.