Agilent rna 6000 nano reagents port 1

SV Total RNA Isolation System (Promega Corporation, Madison, WI, USA) was used to isolate the B. odoriphaga eggs, second instar larvae, fourth instar larvae, pupae, and adult total RNA according to the manufacturer’s protocols, after which possible residual genomic DNA was removed with deoxyribonuclease (DNase I: Fermentas Inc., Burlington, ON, Canada). The RNA integrity was determined using Agilent RNA 6000 Nano Reagents Port 1 (Santa Clara, CA, USA) on a Agilent 2100 Bioanalyzer (Santa Clara, CA, USA) (Table S1). Total RNA from different developmental stages was mixed as one sample for cDNA library construction. cDNA synthesis and library construction was conducted using a Clontech SMARTer PCR cDNA Synthesis Kit (catalogue number: 634925) (Clontech, Mountain View, CA, USA). Sequencing was conducted on the Pacific Biosciences RS II platform (Pacific Biosciences, Menlo Park, CA, USA). BluePippin (Sage Science, Beverly, MA, USA) was used for selection of cDNA sequences ranging in size from 1 to 2 kb, 2 to 3 kb, and 3 to 6 kb. Three, three, and two SMRT (Single-molecule real-time sequencing) cells were used to sequence the 1–2 kb, 2–3 kb, and 3–6 kb libraries, respectively, and the reads were deposited in the NCBI (National Center for Biotechnology Information) Sequence Read Archive database under the accession numbers SRR8903502, SRR8903501, and SRR8903503.