RNA-Seq of venom gland from Sinopoda pengi
Total RNA is used to construction of RNA-seq libraries: mRNA was enriched from the total RNA using oligo (dT) magnetic beads. The enriched RNAs were fragmented into small pieces using divalent cations under 85. Then the cleaved RNA fragments were reverse-transcribed to create the cDNA, which were next used to synthesise U-labeled second-stranded DNAs with E. coli DNA polymerase I. Preparing them for ligation to the indexed adapters; each adapter contains a T-base overhang for ligating the adapter to the A-tailed fragmented DNA. Single-or dual-index adapters are ligated to the fragments, and size selection was performed with AMPureXP beads. The ligated products are amplified with PCR by the following conditions: initial denaturation at 98C for 30 min; 14 cycles of denaturation at 98C for 15 sec, a nealing at 60C for 30sec, and extension at 72C for 30 sec; and then final extension at 72C for 5 min. The average insert size for the final cDNA library was 350 bp (50 bp).