title: RNA-Seq of C. bayani_venom duct
design: Transcriptome sequencing libraries were prepared using NEBNext Ultra directional RNA library prep kit (New England BioLabs, MA, USA) at Genotypic Technology Pvt. Ltd., Bangalore, India. 200 ng of total RNA was taken for mRNA isolation by using NEBNextPoly(A) mRNA magnetic isolation module as per the manufacturers protocol. The isolated mRNA was subjected to fragmentation using magnesium ions at 94C for 8 minutes. The fragmented mRNA was subjected to random priming based reverse transcription in the presence of Actinomycin D (Gibco, Life Technologies, CA, USA) followed by second strand cDNA synthesis. The double stranded cDNA was purified using HighPrep magnetic beads (Magbio Genomics Inc, USA). Purified cDNA was end-repaired, adenylated and ligated to Illumina adapters as per NEBNext protocol. Adapter ligated cDNA was purified using HighPrep beads and subjected to 14 cyclesof enrichment PCR in the presence of specific barcode primer. Final libraries were purified using HighPrep magnetic beads and reconstituted in nuclease-free water. The libraries were quantified by QubitdsDNA HS assay (Thermo Fisher Scientific, MA, USA) and its fragment size distribution was analyzed on Agilent 2200 TapeStation (Agilent Technologies, Santa Clara, USA). The libraries were sequenced by using IlluminaNextSeq 500 sequencer (Illumina, San Diego, USA) for 150 bp paired-end chemistry following manufacturers procedure.