Editing Template swiching cDNA library construction (section)

== First strand cDNA synthesis ==



First strand cDNA synthesis was performed in 106 μl reactions assembled in the following manner. For each sample, the following components were placed in a 200 μl PCR tube on ice: 52.5 μl RNA sample, 8.4 μl oligo dT primer (10 μM), 8.4 μl TS oligo or iso3TS oligo (10 μM), 69.3 μl total volume. RNA and primer mixes were denatured at 65°C for 2 min and then removed to ice. The following reagents were then added to each reaction tube: 20 μl 5× first strand buffer (Powerscript [Clontech] RT buffer: 250 mM Tris (pH 8.3), 30 mM MgCl2, 375 mM KCl, 20 mM DTT), 2 μl DTT (100 mM), 10 μl 50× dNTP (10 mM), 5 μl SuperScript II (Invitrogen, Carlsbad, CA). First strand reactions were incubated at 42°C for 2 hr.