title: Aname sp. (JCUF1) venom gland
design: For RNA extraction, tissue was weighed and homogenised in Trizol at 1mL per 50 mg of tissueand transferred to an RNase-free tube, adding 0.5 mL cold 2-propanol per mL of Trizol. Thesamples were left standing for 5 min, then they were centrifuged at 12,000 g for 10 min at 4C.After removing the supernatant, the pellet was washed with 1 mL 75% ethanol per 1 mL Trizolused in sample preparation. The sample was then centrifuged for 5 min at 7,5000 g at 4C, andethanol was removed and briefly air dried for 5 min. 30 L ultrapure RNase-free water wasadded to the pellet and mixed by pipetting. The resulting RNA extract was stored at -80C forlater use. Total RNA was purified using an RNAClean XP Kit (Beckman Coulter, USA)according to the manufacturers instructions, and RNA sequencing was performed by theSequencing Facility at the UQ Institute for Molecular Bioscience.17Libraries were sequenced as part of a high output run on a HiSeq 2500 (Illumina, San Diego,CA, USA) and RNA-Seq libraries generated using the Illumina Stranded mRNA Library PrepLigation kit (Illumina, 20040534) and IDT for Illumina RNA UD Indexes (Illumina,20040553) according to the manufacturers instructions. 80200 ng of total RNA was enrichedfor mRNA using an oligo-dT bead isolation method, and the mRNA was fragmented in a heatfragmentation step. cDNA was synthesised from fragmented RNA using random primers, andfirst-strand cDNA converted into dsDNA in the presence of dUTP to prevent subsequentamplification of the second strand and thus maintaining the strand orientation of the originalmRNA. The 3 ends of the cDNA were adenylated and pre-index anchors were ligated. Thelibraries were then amplified with 13 cycles of PCR incorporating unique indexes for eachsample to produce libraries ready for sequencing, quantified on the Perkin Elmer LabChip GXTouch with the DNA High Sensitivity Reagent kit (Perkin Elmer, CLS760672), and pooled inequimolar ratios.Raw reads were assembled de novo using an automated pipeline for trimming, correcting, andassembling RNA-Seq reads using fqtrim (https://github.com/gpertea/fqtrim), prinseq(Schmieder and Edwards 2011) and Trinity 2.4.0 (Haas et al. 2013). Trinity's TransDecodersoftware was then used to translate open reading frames >90 bp. These amino acid sequences,together with a list of 200 common MS contaminants, were used as a search database forcomparison to mass spectra obtained from venom. Read mapping was checked and consensussequences corrected manually using Geneious software by Dr. Andrew Walker (IMB, UQ).