RNA-Seq of Azemiops feae: adult venom gland
Total RNA was isolated from the tissue sample by TRIzol kit (Invitrogen, US) and used for ds cDNA synthesis using SMART approach (Zhu et al., 2001). SMART prepared amplified cDNA was amplification by PCR.cDNA synthesis and cDNA amplification.Primer annealing mixture (5 ul) contained: 0.3 g of total RNA; 10 pmol of SMART Oligo II oligonucleotide (5-AAGCAGTGGTATCAACGCAGAGTACGCrGrGrG-3) and 10 pmol of CDS-T22 primer (5-AAGCAGTGGTATCAACGCAGAGTTTTTGTTTTTTTCTTTTTTTTTTVN-3) was composed and heated at 72C for 2 min and cooled on ice for 2 min.  First-strand cDNA synthesis was then initiated by mixing the annealed primer-RNA with Reverse Transcriptase in a final volume of 10 l, containing 1X First-Strand Buffer (50 mM Tris-HCl (pH 8.3); 75 mM KCl; 6 mM MgCl2); 2 mM DTT and 1 mM of each dNTP. The first-strand cDNA synthesis reaction was incubated at 42C for 2 hours in an air incubator and then cooled on ice. First-strand cDNA was diluted 5 times with TE buffer, heated at 70C for 7 min and used foramplification by Long-Distance PCR (Barnes, 1994).PCR reaction (50 l) contained:1 l diluted first-strand cDNA; 1 l 50x Advantage reaction buffer (Clontech, US); 200 M dNTPs; 0.3 M SMART PCR primer (5-AAGCAGTGGTATCAACGCAGAGT-3); 1 l 50x Advantage Polymerize mix (Clontech, US). PCR was performed on MJ Research PTC-200 DNA Thermal Cycler under following program: 95C -7 seconds, 65C -20 seconds, 72C -3 minutes, 17 cycles. Amplified cDNA PCR product was purified using QIAquick PCR Purification Kit (QIAGEN, US).Illumina sequencingWe used the standard Illumina protocol to prepare libraries for DNA using TruSeq Illumina DNA sample preparation kit. Next generation sequencing (NGS) was performed on Illumina Genome Analyzer IIx in the paired-end sequencing mode (2x72 bp)