sRNA-seq of Amblyomma testudinarium: two adult female ticks
Two living ticks were immediately frozen with liquid nitrogen -196°C and preserved at -80°C for 10 days before the RNA extraction using InvitrogenTM Trizol Reagent (Thermo Fisher Scientific, USA). After that, 3 μg total RNA was selected to construct one small RNA library using NEBNext Multiplex Small RNA Library Prep Set for Illumina (NEB, USA) following the manufacturer’s instructions. Briefly, 3' single-stranded adapters were directly and specifically ligated to 3' end of the total RNA without size selection. After the 3' ligation reaction, RT Primers were hybridized to the ligated RNA and the excess of 3' adapters, which transformed the single-stranded DNA adapters into double-stranded DNA molecules. This step is important to prevent adaptor-dimer formation, besides, dsDNAs are not substrates for ligation mediated by T4 RNA Ligase 1 and therefore cannot ligate to 5´single-stranded adapters in the subsequent step. 5´adapters were ligated to 5´ends of the total RNA. Then, cDNAs were synthesized using M-MuLV Reverse Transcriptase with the reduced RNase H activity. PCR amplification was performed using LongAmp Taq 2X Master Mix and PCR Primers with the index "AGTGGACA" for Illumina sequencers. PCR products were purified on 8% polyacrylamide gel (100V, 80 min). DNA fragments in the bright gel band (estimated 130~160 bp) were recovered and dissolved in 8 μL elution buffer.