First-Strand Synthesis Detailed Protocol

First-strand synthesis was performed in a similar manner as described in Parkhomchuk et al (2009) (link). For first-strand synthesis, 8 μl fragmented mRNAs were mixed with 1 μl of random hexamers (Invitrogen) and 1 μl of 10 mM dNTPs. The samples were incubated at 65°C for 5 min and chilled on ice for 1 min. We added 10 μl of a master mix with final concentrations of 1× RT Buffer (Thermo Fisher Scientific), 10 mM MgCl₂, 20 mM DTT, 4 U/μl RnaseOUT (Thermo Fisher Scientific), and 20 U/μl of SuperScript III RT (Thermo Fisher Scientific) to the RNA samples. The samples were then first incubated at 25°C for 10 min, followed by a 50-min incubation at 50°C. The reaction was stopped by incubating samples at 75°C for 15 min. For dNTP cleanup, 80 μl water, 1 μl glycogen, 10 μl 3 M NaOAc (pH 5.2), and 200 μl cold ethanol were added to the samples. Samples were stored at −80°C for 3–7 d. Samples were centrifuged at 14,000 rpm (Eppendorf Centrifuge 5424) for 20 min at 4°C. Supernatant was removed and 500 μl of cold 75% ethanol was added to the samples. Samples were centrifuged again at 14,000 rpm (Eppendorf Centrifuge 5424) for 10 min at 4°C. Supernatant was removed and samples were resuspended in a mixture composed of 51 μl RNAse-free water, 1 μl of 10× RT Buffer, 1 μl 100 mM DTT, 2 μl of 25 mM MgCl₂. Second-strand synthesis was performed as described in Parkhomchuk et al (2009) (link).