Full-length cDNA probe preparation
The workflow of producing full-length cDNA probes is illustrated in Figure 1b. We extracted high-quality RNA from fresh liver tissue of Ptyas korros , a common colubrid species, using the RNA prep Pure Tissue Kit (Tiangen, Beijing). The quality of total RNA was assayed using an Agilent Bioanalyzer 2100 and the RNA integrity number (RIN) was greater than seven. The synthesis of full-length cDNAs was performed using SMARTer™ PCR cDNA Synthesis Kit (Clontech Inc.) based on the manufacturer’s protocols. 4.5 µl of cDNA synthesis mixture containing 1 µg total RNA and 2.67 μM universal tail primer I (included in the kit) was incubated for 3 min at 72 °C and 2 min at 42 °C. The volume was then adjusted to 10 µl with the following reagents: 1× First-Strand Buffer, 2.5 mM DTT, 1mM dNTP Mix, 1.2 μM SMARTer II A Oligonucleotide (included in the kit), 0.25 µl RNase Inhibitor and 10 U SMARTScribe Reverse Transcriptase. Tailing, template switching, and extension were carried out for 90 min at 42°C. The reaction was terminated for 10 minutes at 72°C. The first-strand synthesis product was diluted with 40 μl 1× TE, and used as templates for cDNA amplification. The SMART technology can ensure the synthesized cDNAs are in full-length, and both ends of the synthesized cDNAs contain universal tail sequences.
The synthesized first-strand cDNAs were amplified with a 5’-biotinylated primer (universal tail primer II; see Appendix S1) to generate full-length cDNA probes (Fig. 1b). The PCR reaction mixture contained 1.25 U HiFi Taq DNA Polymerase (TransGen, Beijing), 1× HiFi PCR buffer, 0.2 mM dNTPs, 0.24 μM of universal tail primer II and one μl of diluted first-strand cDNA synthesis product in a total volume of 100 μl. The thermal cycling program is as follows: an initial denaturation for 1 min at 95 °C followed by 19 cycles of 15 s at 95°C, 30 s at 65°C, 6 min at 68 °C. The amplification product was purified by AMpure XP beads and checked on a 1.2% TAE agarose gel. After that, the purified amplification product (full-length cDNA probes) was measured using an ND-2000 spectrophotometer and diluted to a concentration of 50 ng/μl with 1× TE. We did not normalize our full-length cDNA probes to decrease the abundance of highly expressed cDNA, but directly used them for subsequent capture experiments.