RNAseq of Vipera kaznakovi venom gland transcriptome
The venom gland tissue was dissected and homogenised under liquid nitrogen using a pestle and mortar and a TissueRuptor (QIAGEN), and total RNA was extracted using the TRIzol Plus RNA Purification System (Thermofisher) protocol. The RNA samples were DNAse treated (On-Column PureLink DNase; Life Technologies) and total RNA eluted in 30L nuclease free water (QIAGEN). Total RNA underwent two rounds of poly(A) selection using the Dynabeads mRNA DIRECT purification kit (Life Technologies), and was then eluted in nuclease free water, before the quality and quantity of isolated RNA was assessed using a Bioanalyzer (Agilent). One L of the resulting total RNA was loaded onto an Agilent RNA 6000 Nano Chip and inserted into the Agilent 5000 Bioanalyzer for analysis. Agilent RNA 2100 Expert software was used to calculate total RNA concentration and assign a RNA integrity number (RIN) (Schroeder et al., 2006). Subsequently, the sequencing library was prepared using 50 ng of enriched RNA using the Script-Seq v2 RNA-Seq Library Preparation Kit (epicenter), following 12 cycles of amplification. The sequencing library was then purified using AMPure XP beads (Agencourt), quantified using the Qubit dsDNA HS Assay kit (Life Technologies) and the size distribution assessed using a Bioanalyser (Agilent). Sequencing libraries were multiplexed with others not reported in this study, and sequencing performed on an Illumina MiSeq platform using 250bp paired-end reads technology (Centre for Genomics Research, University of Liverpool). The Vipera kaznakovi sample represented 1/6th of this sequencing lane.