Second strand master mix

Preparation of cDNA libraries was performed following the Illumina TruSeq^® RNA Sample Preparation v2 Guide. Briefly, both polyA-RNAs and v-RNAs were fragmented to sizes of 120 to 210 nt at 94°C for 5 min. The short fragments were used as templates for synthesizing first-strand cDNA using SuperScript II reverse transcriptase (RTase) (Invitrogen^TM) and random hexamer primers (Illumina) at the condition of 25°C for 10 min, followed by 42°C for 50 min for synthesis and 70°C for 15 min for RTase inactivation. The second strand cDNA was subsequently synthesized using Second Strand Master Mix (Illumina) at 16°C for 1 h. Subsequently, a poly(A) adaptor was added at the 3' end of the resulting short cDNA fragments, followed by ligation of sequencing adaptors using Illumina’s adaptor oligo mix. The 200-bp-long fragments were purified through gel extraction and were further enriched according to the instructions of the Illumina TruSeq^® RNA Sample Preparation Kit v2 to prepare the final cDNA library. Sequencing was conducted at Genetech Biotech Co., Ltd. (Taipei, Taiwan) using an Illumina MiSeq system. The polyA-RNA template was employed to synthesize the paired-end reads, and single-end reads were obtained from the v-RNA template.

Samples were homogenized at 6,500 rpm for 2 × 20 s in a Precellys 24 homogenizer (Bertin Instruments, France). Total RNA was extracted from whole larvae following the QIAzol protocol (Qiagen, Germany). RNA concentration, purity and quality were determined using the NanoDrop 1000 (Thermo Scientific, USA) and the TapeStation 2200 (Agilent Technologies, USA).
TruSeq libraries were prepared from total RNA according to the manufacturer’s protocol (Illumina, USA). After purification with oligo-dT beads, mRNAs were washed and fragmented into an average length of 508–541 base pairs. The first strand of complementary DNA was synthesized with random hexamer primers (Illumina, USA), while the second strand was synthesized by Second Strand Master Mix (Illumina, USA). All 18 libraries were barcoded and normalized with the KAPA library quantification kit (Kapa Biosystems, USA). The pooled libraries were then denatured according to the NextSeq System Denature and Dilute Libraries Guide (Illumina, USA) and loaded at 11 pM on a NextSeq 500 reagent cartridge (Illumina, USA) for 150 cycle, paired-end sequencing at the Nord University genomics platform (Norway).

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.