RNA-seq of Dispholidus typus: venom gland tissue from a single adult specimen
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 ľg of the resulting RNA enriched 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 5Ő and 3Ő 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).