RNA-Seq of Plateau brown frog:adult liver
The NEB Next Ultra II RNA Library Prep Kit for Illumina (New England Biolabs, USA) was used for library construction. mRNA with poly(A) tails was enriched using Oligo(dT) magnetic beads (Invitrogen, USA) followed by random fragmentation of mRNA using divalent cations. Using fragmented mRNA as templates, cDNA was synthesized with random oligonucleotides as primers. Double-stranded cDNA was purified, followed by end repair and 3' end adenylation with the addition of "A" bases and ligation of sequencing adapters. cDNA fragments of approximately 400-500 bp were size-selected using AMPure XP beads (Beckman, USA), subjected to PCR amplification, and purified again using AMPure XP beads to obtain the final library. Library quality was assessed using the Agilent 2100 Bioanalyzer (Agilent, USA) with the Agilent High Sensitivity DNA Kit (Agilent, USA). The total library concentration was determined using the PicoGreen assay (Quantifluor-ST fluorometer, Promega, USA; Quant-iT PicoGreen dsDNA Assay Kit, Invitrogen, USA), and the effective library concentration was quantified using QPCR (Thermo, USA). After normalization of multiplexed DNA libraries, samples were mixed in equal volumes. Finally, the mixed library was diluted stepwise for quantification and subjected to PE150 sequencing on an Illumina sequencing platform (Illumina, USA).