Pe adapter oligo mix

RNA sequencing libraries were constructed in parallel from the six species using TruSeq RNA Sample Prep Kits (Illumina, SanDiego, USA). Briefly, first strand cDNA synthesis was performed with oligo-dT primer and Superscript II reverse transcriptase (Invitrogen, CA, USA). The second strand was synthesized with Escherichia coli DNA Pol I (Invitrogen, CA, USA). Double-stranded cDNA was purified with a Qiaquick PCR purification kit (Qiagen), and sheared with a nebulizer (Invitrogen, CA, USA) into 200–250 bp fragments. After the end repair and addition of a 3′-dA overhang, the cDNA was ligated to Illumina PE adapter oligo mix (Illumina). The products were then purified and enriched with PCR to create the final sequencing cDNA library. Both ends of the library were sequenced on the Illumina HiSeq 2000 platform.

3 μg of total RNA per sample was used as input material for the RNA sample preparation. Beads with oligo (dT) were used to isolate poly(A) mRNA from total RNA. RNA sequencing libraries were constructed from these mRNA using the TruSeq RNA Sample Preparation Kit (Illumina, San Diego, USA). Briefly, the Elution 2-Frag-Prime (94 °C for 8 minutes, 4 °C hold) was used to elute, fragment and prime the mRNA with Elute, Prime, Fragment Mix (Illumina). First strand cDNA synthesis was performed with First Strand Master Mix and SuperScript II mix (ratio: 1ul SuperScript II/7ul First Strand Master Mix) (Invitrogen). The second strand was synthesized with Second Strand Master Mix (Illumina) and Ampure XP beads (Illumina) were used to separate the double-stranded (ds) cDNA from the 2nd strand reaction mix. After end repair and the addition of a 3′-dA overhang, the cDNA was ligated to Illumina PE adapter oligo mix (Illumina), and size-selected for 350 ± 20 bp fragments by gel purification. After 15 cycles of PCR amplification, the 350 bp paired-end libraries were sequenced using the paired-end sequencing module (100 bp at each end) of the Illumina HiSeq 2000 platform.