title: RNAseq of Aspidelaps scutatus intermedius venom gland transcriptome
design: Total RNA was extracted using the TRIzol Plus RNA Purification kit (Life Technologies, Carlsbad, CA, USA). The RNA sample was then DNAse treated (Ambion TURBO DNase, Invitrogen) and 1 g of the resulting RNA enricVenom gland transcriptome sequencing reads from the snake Aspidelaps sucutatus intermedius. 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 A. s. intermedius sample represented 1/6th of this sequencing lane and resulted in 5,664,906 reads. The ensuing read data was quality processed, resulting in the removal of adapter sequences (Cutadapt; https://code.google.com/p/cutadapt/) and low quality bases (Sickle; https://github.com/najoshi/sickle).hed using a polyA selection method (Dynabeads mRNA Purification Kit from total RNA preps, ThermoFisher Scientific). The RNA-Seq library was prepared from 50 ng of the enriched RNA material using the TruSeq Stranded mRNA HT Sample Prep Kit (Illumina). During the preparation, dual index adapters were used so that other RNA samples in the sequencing pool (not described here) had a unique index at both the 5 and 3 ends. The sequencing library was amplified with 12 cycles PCR and then purified using AMPure XP beads (Agencourt, Brea, CA, USA), quantified using the Qubit dsDNA HS Assay Kit (Life Technologies) and the size distribution assessed using a Bioanalyser (Agilent). The quantity and quality of the resulting sample was also assessed by qPCR using the Illumina Library Quantification Kit on a Roche Light Cycler LC480II according to manufacturer's instructions. Finally, the template DNA was denatured according to the protocol described in the Illumina cBot User guide and loaded at 10 pM concentration. Sequencing was carried out on a single lane of an Illumina MiSeq with 2x250 bp paired-end sequencing and v2 chemistry (Centre for Genomic Research, University of Liverpool, UK).