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Old 11-27-2013, 06:29 AM   #21
TonyBrooks
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Location: London

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You digest the second strand cDNA before PCR using an enzyme that cleaves wherever there is a uracil base (dUTP is used in place of dTTP in the second strand mastermix). This means when you do the PCR you are only amplifying from the first strand cDNA, hence all reads will from the same strand as the mRNA. The first strand cDNA will be complementary to the mRNA that produced it. The PCR product is still double-stranded
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