*2.4. Sequencing of mRNA, Gene Ontology, and Pathway Analysis*

Construction of a library of RNAs was performed using the SENSE mRNA-Seq Library Prep Kit (Lexogen, Inc., Vienna, Austria). Briefly, 2 μg of total RNA was processed and incubated with oligo-dT magnetic beads, after which other RNAs except mRNA were eliminated with a washing solution. Random hybridization of starter/stopper heterodimers was applied to the poly(A)RNA still bound to the magnetic beads in order to produce libraries. These heterodimers consisted of Illumina-compatible linker sequences. A single-tube reverse transcription and ligation reaction was applied to extend the starter to the next hybridized heterodimer. Then, the newly synthesized cDNA insert was bound with the stopper. The release of the library from the beads was done by second-strand synthesis. The library was amplified afterward and bar codes were introduced. High-throughput sequencing was done using HiSeq 2500 (Illumina, San Diego, CA, USA) as paired-end 100 bp sequencing.

Software tools (TopHat, Toronto, ON, Canada) were used to map RNA-Seq reads. Transcript assembly and detection of differentially expressed genes or isoforms were performed from the alignment file using cufflinks [19]. The quantile normalization method was used for comparison between samples [20]. Functional gene classification was done using Medline databases (http://www.ncbi.nlm.nih.gov/), DAVID (http://david.abcc.ncifcrf.gov/), GenMAPP (http: //www.genmapp.org/), and BioCarta (http://www.biocarta.com/) [21]. Pathway analysis was performed on differentially expressed genes [22]. A fold-change of 1.3 and a log2-normalized read count of 4 were the thresholds applied for this study [23].
