RNA-Seq of Thamnophis saurita: Venom Gland
The RNA extraction process followed a standard Trizol extraction procedure (Rokyta et al. 2013; Hofmann et al. 2018; Strickland et al. 2018). We extracted RNA from each individuals left and right Duvernoys gland and then combined RNA from each gland in equal concentration to create comprehensive Duvernoys gland RNA pools for each individual. Using a Qubit RNA Assay (Thermo Fisher Scientific), RNA was quantified and quality checked on a BioAnalyzer RNA Pico chip or a 4150 TapeStation System (Agilent Technologies, Santa Clara, CA, USA). Before the RNA extracts were ready for sequencing, cDNA libraries had to be prepared. For this cDNA library preparation, we used the NEBNext Ultra II RNA Library Prep Kit (E7775) with the Ploy(A) mRNA Magnetic Isolation Module (E7490). After mRNA isolation and cDNA synthesis, we targeted RNA inserts with a size of 300bp by using a fragmentation incubation time of 15 minutes and 15 PCR cycles to yield the desired cDNA concentration. Purification of cDNA samples was done using NEBNext Sample Purification Beads. A Qubit DNA BR Assay (Thermo Fisher Scientific) was used to determine DNA concentration. Quality control of samples was done using a Bioanalyzer with a HS DNA kit according to the manufacturers instructions or a 4150 TapeStation System (Agilent Technologies, Santa Clara, CA, USA). Libraries were sequenced with Illumina SBS technology at Florida State University or Clemson University.