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PCR conditions were 95C for 3 min, then 40 cycles consisting of denaturation at 95C for 10 s, annealing at 60C for 10s and extension at 72C for 30 s, followed by the melting curve protocol with 10s at 95C and then 60s each at 0.5C increments between 65C and 95C

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The digested peptides were reconstituted in 2% acetonitrile/0.1% TFA and 5 l of each sample was loaded onto a PepMap 100 3U 75 m 20 mm reverse-phase trap where they were desalted online before being separated on a 100 2U 50-micron 150 mm PepMap EasySpray reverse-phase column 17

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