Rana arvalis class I AMP targeted sequencing, TP3 primer
First strand cDNA was synthesized from 2 microg of total RNA with qScriptTM Flex cDNA Synthesis Kit (Quanta Biosciences, USA) using 4 microl of qScriptTM Flex Reaction Mix, 1 microl of qScriptTM Reverse Transcriptase and 1 microl of poly-dT in a total volume of 20 microl. Mixture for cDNA amplification contained 0.2 microl of 5 U/microl DNA polymerase, 2.5 microl of MgCl2, 2.5 microl of buffer A (KAPA Taq PCR Kit, Kapa Biosystems, USA) together with 0.5 microl of 10 mM dNTPs, 0.5 microl of 100 microM each specific primer and 1 microl of template in total volume of 20 microl. PCR program started with initial denaturation at 95°C for 2 min, followed by 10 cycles including 10 sec at 95°C, annealing at 45-50°C (0.5°C/cycle) and 20 sec elongation at 72°C. For the next 25 cycles annealing temperature was set to 52 °C for 20 sec ending with 5 min final elongation at 72°C.   RT-PCR amplification (switch PCR) was performed to attach the barcodes, a sample-specific 10 nucleotide sequence used for de-multiplexing, on 5' end of the amplicon, which were followed by the adapters. Mixture for RT-PCR contained 0.2 microl of 5 U/microl DNA polymerase, 2.5 microl of buffer A (KAPA Taq PCR Kit, Kapa Biosystems, USA) together with 1 microl EvaGreenTM (Biotium, USA), 0.5 microl of 10 mM dNTPs, 1 microl of template and 3 l of primers in total volume of 20 microl. RT-PCR program was performed for 8 cycles including denaturation at 95°C for 10 sec, annealing at 60°C for 10 sec and 40 sec elongation at 65°C with 3 min final elongation at 72°C. The quality of the amplification products was visualized by electrophoresis migration on 1.5 % agarose gel after each amplification step. Based on this analysis some libraries were discarded due to unsuccessful PCR or out of range amplicon size.  Size range and quantification of each individual library were assessed with a DNA 1000 kit on Agilent Bioanalyzer 2100 (Agilent Technologies, USA)
University of Trieste
Rarv_TP3