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.