T easy vector

In this study, R. microplus ticks were collected from Yongjing County, Gansu Province, and maintained under laboratory conditions. These ticks were identified using morphology by the Animal Research Institute (Lanzhou Veterinary Research Institute). Approximately 0.5 mg of eggs and 1 g of larvae were used for miRNA extraction, and another 1 g of larvae was placed in a cloth bag attached to cattle for 25 ± 3 days to produce engorged adult female ticks. To analyze the expression profile of miRNAs in R. microplus, unfed larvae, adult female ticks and eggs were ground in liquid nitrogen to extract total RNA using TRIzol reagent (Invitrogen, Cat No. 15596-026). The concentration of RNA was 400 ng/mL (260/280 = 2.00). Some RNAs were used to construct libraries for small RNA sequencing, and some were used for miRNA validation. The synthesis of first-strand cDNAs was performed according to the protocol for transcriptase XL (Avian Myeloblastosis Virus, AMV) (TaKaRa, Shiga, Japan) with a loop primer of miRNAs and oligo dT18. PCR was performed to obtain the miRNAs sequence. The PCR product was ligated to the T-Easy vector (TaKaRa, Japan), which was transformed into JM109 (TaKaRa, Japan). The sequence was obtained by GenScript (Nanjing, China).

Purified PCR products were ligated to the T-easy vector (Takara Bio Inc.). The ligation mixture was prepared with 2 µL ligation buffer, 3 µL T-easy vector, 1 µL T4 ligase, and 4 µL PCR water. Recombinant plasmids were transformed into Escherichia coli JM109 (DE3) (Promega, USA), cultured on Luria-Bertani agar plates supplemented with ampicillin (LB amp+) and incubated overnight at 37 °C. Subsequently, LB broth (4 mL) was inoculated with the grown colonies and incubated overnight at 37 °C, following which plasmids were extracted using the AccuPrep^® plasmid extraction kit (Bioneer). Extracted plasmids were sent for sequencing at Macrogen Pvt. Ltd. (South Korea).

Total RNA from exponentially growing cells in LB medium was extracted with total RNA extraction reagent (Vazyme) as recommended by the manufacturer. Following extraction, total RNA was treated with DNaseI, which was later heat‐inactivated at 70°C for 15 min. The transcriptional start site was determined using the 5′ rapid amplification of cDNA ends (5′ RACE) system, as recommended by the supplier (Clontech, www.bdbiosciences.com) with 3 μg of total RNA (DNA‐free) obtained above. The gene‐specific primer bifA‐race (Table 1) was used to initiate the first‐strand cDNA synthesis. Small aliquots of cDNA as templates were amplified using SMART PCR primer mix, UP, and gene‐specific primer bifA‐race (Table 1). The resulting PCR product was cloned into the T‐easy vector (Takara) according to the manufacturer's instructions before being sequenced.