2.1.5. Library Construction and RNA Sequencing

Approximately 1 μg of RNA from each sample was used to generate RNA-Seq cDNA libraries for sequencing using the TruSeq RNA Sample Prep Kit v2 (Illumina, Inc., San Diego, CA, USA). Sample preparation followed the manufacturer's protocol with a workflow that included isolation of polyadenylated RNA molecules using poly-T oligo-attached magnetic beads, enzymatic RNA fragmentation, cDNA synthesis, ligation of bar-coded adapters, and PCR amplification. Ambion External RNA Controls Consortium (ERCC) RNA Spike-In Control Mix 1 (Life Technologies Corporation, Carlsbad, CA, USA) was added to the samples. The amplified cDNA fragments were sequenced using a HiSeq 2000 sequencing system (Illumina, San Diego, CA, USA). Finally, sequencing data were converted to raw data in FASTQ format utilizing illumina package bcl2fastq.
