RNA-Seq of Macrothele yani: adult female venom glands
Oligo(dT) attached magnetic beads were used to purifed mRNA from venom gland in three replicates by eliminating rRNA and tRNA in a total amount of 2 g RNA per sample. Purifed mRNA was fragmented into small pieces with fragment bufer at appropriate temperature. Then, First-strand cDNA was generated by random hexamer-primed reverse transcription, followed by a second-strand cDNA synthesis. Afer that, RNA Index Adapters and A-Tailing Mix were added by incubating to end repair. The fragments of cDNA obtained from previous steps were amplifed by PCR, and the PCR products were purifed by Ampure XP Beads, and dissolved in EBsolution. Te products were validated on the Agilent Technologies 2100 bioanalyzer for quality control. The double stranded PCR products obtained from previous steps were denatured by heating and circularized by the splint oligo sequence to get the fnal library, and the single stranded circular DNA (ssCir DNA) was formatted as the fnal library. The constructional fnal libraries were further amplifed with phi29 to make DNA nanoball (DNB), DNBs were loaded into the patterned nano-array and paired-end of150 bp base reads were generated on T7 platform by Wuhan Benagen Technology Co., Ltd.