RNA-Seq of Odorrana grahami: adult male skin (CRX1021362; CRA015896)
Total skin RNA was extracted using a Trizol reagent as per the manufacturer's protocol. Purification of mRNA was performed using poly-T oligo-attached magnetic beads, followed by fragmentation. First-strand cDNA was synthesized using M-MuLV Reverse Transcriptase and random hexamer primers, and RNaseH was used to degrade the RNA. Subsequently, second-strand cDNA synthesis was conducted using DNA Polymerase I and dNTP. The remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. Adenylation of 3' ends of DNA fragments was performed, and an adaptor with a hairpin loop structure was ligated to prepare for hybridization. The library fragments were purified using the AMPure XP system (Beckman Coulter, Beverly, USA). Following PCR amplification, the PCR product was purified by AMPure XP beads, and the library was finally obtained. The library was initially quantified using a Qubit2.0 Fluorometer (Invitrogen, Carlsbad, CA, USA), and the insert size of the library was detected using an Agilent 2100 bioanalyzer. Finally, qRT-PCR was used to accurately quantify the effective concentration of the library. The library preparations were sequenced on an Illumina NovaSeq 6000 with a paired-end sequencing length of 150 bp (PE150) by Novogene (Beijing, China).