by diluting to the desired distillate concentration. The concentrations and treatment conditions for the pollutants were adopted from the studies assessing the toxic effects of toluene and formaldehyde in Drosophila. The used concentrations of pollutants demonstrated the low toxicity in Drosophila. However were much higher than maximum allowable concentrations. For example, 
according the Federal Drinking Water Standards the MAC for TCDD and toluene are 0.93161027 mM/ L and 10.85 mM/L, respectively. Thus the used concentrations of TCDD in 16107 and toluene in 4.66103 times exceeded the MAC. According to the Occupational Safety and Health Administration, permissible exposure limits for occupational formaldehyde exposure are 0.75 ppm at or below an 8-hour time-weighted average and the short-term exposure limit of 2 ppm. However we did not estimate the concentration of formaldehyde inside the vial. The data from literature suggest that several hours of exposure is sufficient for the induction of changes in gene expression. At the same time, the extra time after exposure can lead to the effects of unaccounted factors. The flies in the control- and experimental groups were fixed in liquid nitrogen immediately after treatment. Integrity Score $8) was determined using an Agilent 2100 Bioanalyzer per each manufacturer’s instructions. mRNA Library Preparation To prepare samples for the mRNA sequencing libraries, we used the Illumina TruSeqTM RNA Sample Preparation Kit . Purification and Fragmentation of PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19653943 mRNA In summary, 2.53.5 mg of total RNA from each sample of Drosophila was used to purify the poly-A containing mRNA molecules by poly-T oligo-attached magnetic beads, with two rounds of purification. During the second elution of poly-A RNA, the RNA was also fragmented and primed for cDNA synthesis according to the manufacturer’s protocol. cDNA Synthesis The fragmented mRNA samples were subjected to cDNA synthesis according to the manufacturer’s protocol. Briefly, cDNA was synthesized from fragmented RNA using a SuperScript Double-Stranded cDNA Synthesis kit. The cDNA was then converted into double-stranded cDNA using the reagents supplied in the kit. Ampure XP beads were used to separate ds cDNA from the second-strand reaction mix. RNA Isolation Total RNA was extracted from 10 Drosophila images with ZR RNA MiniPrepTM per the manufacturer’s instructions. The RNA quantity was determined using a QubitH 2.0 Fluorometer and the RNA integrity and females. The color scale indicates the number of genes in a group. doi:10.1371/journal.pone.0086051.g003 Preparation of cDNA Library The double-stranded cDNA was subjected to library preparation using the Illumina TruSeqTM RNA sample preparation kit according to the manufacturer’s protocol. The ds cDNA was blunt-ended through an end-repair reaction. Next, a single `A’ LBH589 manufacturer nucleotide was added to the 39 ends of the blunt fragments to prevent them from ligating to one another during the adapter ligation reaction. The multiple indexing adapters contain a single `T’ nucleotide on the 39 end that provides a complementary overhang for ligating the adapter to the fragment. The cDNA fragments were then ligated to specific RNA Adapter Indexes supplied in the kit. The In-Line Control DNA was added to each enzymatic reaction. The controls contain ds DNA fragments designed to indicate the success or failure of specific enzymatic activity used in the library preparation process. Library Validation The quantity of libraries wa
