Transtaq hifi buffer 2

Total RNA was extracted as described above, and the cDNA was synthesized from 1 μg RNA using PrimeScript^® RT reagent Kit With gDNA Eraser (TaKaRa, Japan). Sixteen pairs of primers for obtaining pre-miRNA sequences were listed in Supplemental Table S6. Polymerase chain reaction (PCR) contained 2.5 μL cDNA, 2 μL mix solution of target gene primers, 2.5 μL 10× TransTaq^® HiFi Buffer II, 2 μL dNTPs (2.5 mM), 0.3 μL TransTaq^® HiFi DNA Polymerase (TransGen, China), 15.7 μL of ddH₂O. And the amplification was performed under the following conditions: 94 °C denaturation for 3 min, 35 running cycles of 94 °C for 30 s, 55.2 °C for 30 s, 72 °C for 30 s, and an elongation cycle of 72 °C for 10 min. PCR products were separated by 3% agarose gel electrophoresis, and the incised gels were purified using a TaKaRa MiniBEST Agarose Gel DNA Extraction Kit Ver.3.0 (TaKaRa, Japan). The extracted products were cloned into pEASY^TM-T5 Zero vector (TransGen, China) and transformed into competent Escherichia coli Trans1-T1 cells (TransGen, China). The recombinant plasmids were sent Shanghai Sangon Biological Engineering Technology & Services Co., Ltd. (Shanghai, China) to sequence.

Seven putative lncRNAs (lncRNA1–7) identified from RNA-seq data were validated using PCR assay (Additional file 9: Table S9). An aliquot of the total RNA used to construct the libraries was reverse-transcribed using a RevertAid First Strand cDNA Synthesis kit (Thermo Scientific™, Waltham, MA) following the manufacturer’s recommendations. Subsequently, the reaction products were diluted 20-fold to serve as the template in a PCR driven by the primer pairs given in Supplementary Table S9. Each 50 μL reaction comprised 5 μL of 10× TransTaq® HiFi Buffer II (Transgen Biotech, Beijing, China), 4 μL of 2.5 mΜ dNTP (each 10 μM), 1 μL of the forward primer and 1 μL of the reverse primer (each 10 μM), 4 μL of the cDNA template (50 ng/μL), 1 μL of TransTaq® HiFi DNA Polymerase (Transgen Biotech), and 35 μL of ddH₂O. The amplified product was purified (TIANgel Midi Purification Kit DP209, TIANGEN Biotech, Beijing, China) and subcloned into the pEASY-T1 Cloning Vector (Transgen Biotech) for Sanger sequencing.