title: RNA-Seq of Oncocephalus sp. venom glands
design: For RNA extraction, venom glands were harvested from one adult bug after anesthesia with CO2 for 5 min. The main gland posterior lobe, main gland anterior lobe, and accessory gland were pooled and stored in >10 the glandular volume of RNAlater (Ambion, Austin, TX). Total RNA was extracted using a DNeasy kit (Qiagen, Mississauga, Canada), and mRNA was isolated using a Dynabeads mRNA Direct kit (Ambion) according to the manufacturer's instructions.RNA sequencing (RNA-Seq) was performed on the NextSeq sequencer (Illumina, San Diego, CA) at the Institute for Molecular Bioscience Sequencing Facility at the University of Queensland. After TruSeq library preparation, each sample was run on four lanes of a 150 cycle mid-output run to generate 150-bp paired-end. For each species, eight assemblies were constructed using CLC Genomics Workbench (CLC Bio, Aarhus, Denmark) and Trinity (35). For CLC assemblies, reads with q-scores below 30 were trimmed and the reads assembled using minimum contig length of 150 bp, minimum similarity to join contig 0.95, and word (k-mer) sizes of 21, 24, 29, 34, 44, 54, and 64. For Trinity, which employs a fixed k-mer method, reads were assembled using the default trimming parameters and minimum contig length of 150 bp. For each gland compartment, contigs from the Trinity and CLC assemblies were pooled and clustered using CD-HIT (threshold 95%) and then re-imported into CLC Genomics Workbench where trimmed reads were re-mapped and used to update the final contigs. Open reading frames (ORFs) of 90 bp and greater were extracted using TransDecoder, to which 155 common LC-MS/MS contaminant sequences were added to produce the final sequence database for searching.