Reverse transcription with MarathonRT

MarathonRT purification was performed as described in (^{Guo et al., 2020}). For each amplicon, 500ng of total cellular RNA was mixed with 1μL of the corresponding 1μM RT primer. Gene-specific primers used for RT are listed in Table S2. Primers were annealed at 65°C for 5min then cooled to room temperature, followed by addition of 8μL of 2.5x MarathonRT SHAPE-Map Buffer (125mM 1M Tris-HCl pH 7.5, 500mM KCl, 12.5mM DTT, 1.25mM dNTPs, 2.5mM Mn²⁺), 4μL of 100% glycerol, and 0.5μL of MarathonRT. RT reactions were incubated at 42°C for 3 hours. 1μL 3M NaOH was added to each reaction and incubated at 95°C for 5min to degrade the RNA, followed by the addition of 1μL 3M HCl to neutralize the reaction. cDNA was purified using AmpureXP beads (Cat. No. A63880) according to manufacturer’s protocol and a 1.8x bead-to-sample ratio. Purified cDNA was eluted in 10μL nuclease-free water.