Min G homodimer, -37.6 cysG: kcal; Min monodimer, -15 G kcal Location of each primer by exon E NA or intron (if applicable) What splice variants are targeted? E NA qPCR oligonucleotides Primer sequences E + See Table S2 RTPrimerDB identification D NA number Probe sequences D NA Location and identity of any E NA modification Manufacturer of oligonucleotides D NA Purification method D NA qPCR protocol Complete reaction conditions E + Reaction volume and amount of Total volume 20 containing 1 μl, E + cDNA/DNA of cDNA as template μl Primer, (probe), Mg2+, and dNTP Primer 10 pM, dNTP 25 mM E + concentration each Polymerase identity and E + Proprietary (Enzynomics) concentration TOPrealTM qPCR 2X PreMIX Buffer/kit identity and E + (SYBR Green with high ROX) manufacturer (Enzynomics, Daejeon, Korea) chemical composition of the D - Proprietary buffer Additives (SYBR Green I, E + SYBR Green I DMSO, and so forth) MicroAmp Fast Optical 96-Well Manufacturer of plates/tubes and D + Reaction Plate, 0.1 mL catalog number (Thermofisher, Cat # : 4346907) Holding stage (95 °C 10 min), cycling stage (95 °C 10 s, 55 °C Complete thermocycling 15 s, 72 °C 20 s (+)), and melt E + parameters curve stage (95 °C 15 s, 1 min, +0.3 °C (+), 95 °C 15 s )(+) Reaction setup (manual/robotic) D + Manual a StepOnePlus Real-time PCR Manufacturer of qPCR instrument E + system (Applied Biosystems, Foster City, CA, USA) qPCR validation Evidence of optimization (from D + From standard curve gradients)
cysG: Min G homodimer, -37.6 kcal; Min G monodimer, -15 kcal Location of each primer by exon or intron (if applicable) E NA What splice variants are targeted? E NA qPCR oligonucleotides Primer sequences E + See Table S2 RTPrimerDB identification number D NA Probe sequences D NA Location and identity of any modification E NA Manufacturer of oligonucleotides D NA Purification method D NA qPCR protocol Complete reaction conditions E + Reaction volume and amount of cDNA/DNA E + Total volume 20 μl, containing 1 μl of cDNA as template Primer, (probe), Mg2+, and dNTP concentration E + Primer 10 pM, dNTP 25 mM each Polymerase identity and concentration E + Proprietary (Enzynomics) Buffer/kit identity and manufacturer E + TOPrealTM qPCR 2X PreMIX (SYBR Green with high ROX) (Enzynomics, Daejeon, Korea) chemical composition of the buffer D - Proprietary Additives (SYBR Green I, DMSO, and so forth) E + SYBR Green I Manufacturer of plates/tubes and catalog number D + MicroAmp Fast Optical 96-Well Reaction Plate, 0.1 mL (Thermofisher, Cat # : 4346907) Complete thermocycling parameters E + Holding stage (95 °C 10 min), cycling stage (95 °C 10 s, 55 °C 15 s, 72 °C 20 s (+)), and melt curve stage (95 °C 15 s, 1 min, +0.3 °C (+), 95 °C 15 s )(+) Reaction setup (manual/robotic) D + Manual Manufacturer of qPCR instrument E + a StepOnePlus Real-time PCR system (Applied Biosystems, Foster City, CA, USA) qPCR validation Evidence of optimization (from gradients) D + From standard curve
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