Instrument: Illumina HiSeq 2000
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: SINGLE
Construction protocol: The fat body and ovary were collected from different females in each group at the following post-treatment time points: (1) the Sephadex group: 4 hours, 6 days; (2) the bacterial parasite group: 4 hours,6 days;(3) the dipteran parasite group: 4 hours,6 days. The RNA was extracted using the Trizol reagent (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instructions. Three micrograms of total RNA was used as the input material for sequencing. All of the samples had RIN values greater than 8.The sequencing library was generated using the TruSeq RNA Sample Preparation Kit (Illumina, San Diego, USA), according to the manufacturer’s recommendations. Four index codes were added to the sequence. The mRNA was purified from the total RNA using poly(T)-oligo-conjugated magnetic beads. Fragmentation was performed by heating the RNA in fragmentation buffer (Illumina) containing divalent cations. First-strand complementary DNA (cDNA) was performed using random oligonucleotides and SuperScript II reverse transcriptase (Invitrogen). Second-strand cDNA synthesis and RNA degradation was performed using DNA Polymerase I and RNase H. The overhangs were converted to blunt ends through the exonuclease activity of the polymerase, and the double-stranded DNA fragments were purified. The 3' ends of the DNA fragments were adenylated, and Illumina PE adapter oligonucleotides were ligated to both termini of the adenylated fragments for hybridization. To select the adaptor-ligated cDNA fragments approximately 200 bp in length, the library fragments were purified using the AMPure XP system (Beckman Coulter, Brea, CA, USA). The purified cDNA fragments were selectively enriched during 10 cycles of polymerase chain reaction (PCR) using the Illumina PCR Primer Cocktail. Products were purified again, and quantified by performing the High-sensitivity DNA Assay (Agilent Technologies, Santa Clara, CA, USA) using the Bioanalyzer 2100 system (Agilent). The clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumina), according to the manufacturer’s instructions.