title: RNA-Seq of Bothrops brazili: venom glands
design: Four days after venom extraction, venom glands were dissected and immediately transferred to RNALater (Invitrogen, Life Technologies Corp.) and stored at -80C. The RNA was extracted based on the use of guanidine isothiocyanate followed by phenolic extraction as previously described by. Initially, the total RNA was extracted with Trizol and quantified by Quant-iT RiboGreen RNA reagent and Kit. Quality control of the extracted RNA was then performed in an Agilent 2100 Bioanalyzer using an Agilent RNA 6000 Nano Kit to verify the integrity of total RNA through band discrimination corresponding to fractions 18S and 28S of total RNA. Libraries were prepared for each individual sample. One microgram of total RNA was used with an Illumina TruSeq Stranded RNA HT kit consisting of TruSeq Stranded RNA HT/cDNA Synthesis PCR, TruSeq Stranded RNA HT/Adapter Plate Box, and TruSeq Stranded HT mRNA. Libraries were validated by considering their fragment size distribution and their quantification. Fragment size distributions were evaluated by microfluidic gel electrophoresis in a Bioanalyzer device (Agilent 2100) using an Agilent DNA 1000 Kit according to the manufacturers protocol. Quantification of each library was then performed by real-time PCR using a KAPA SYBR FAST Universal qPCR Kit, according to the manufacturers protocol, using the StepOnePlus Real-Time PCR System. Aliquots of each cDNA library were diluted to a concentration of 2 nM. Next, a pool of all samples, 5 l of each library, was prepared and the concentration of the pool was again determined by real-time PCR. The cDNA libraries were sequenced on an Illumina HiSeq 1500 System in Rapid Run mode using a paired-end flow cell for 300 cycles of 2*150 bp.