Illustra g 50 microspin columns

After SHAPE and RNP-MaP RNA modification and purification, MaP cDNA synthesis was performed using a revised protocol as described (Mustoe et al., 2019). Briefly, 7 μl of purified modified RNA was mixed with 200 ng of random 9-mer primers and 20 nmol of dNTPs and incubated at 65°C for 10 min followed by 4°C for 2 min. 9 μl 2.2 2′-MaP buffer [1′ MaP buffer consists of 6 mM MnCl₂, 1 M betaine, 50 mM Tris (pH 8.0), 75 mM KCl, 10 mM DTT] was added and the combined solution was incubated at 23°C for 2 min. 1 μl Superscript II Reverse Transcriptase (200 units, Invitrogen) was added and the reverse transcription (RT) reaction was performed according to the following temperature program: 25°C for 10 min, 42°C for 90 min, 10′ [50°C for 2 min, 42°C for 2 min], 72°C for 10 min. RT cDNA products were then purified (Illustra G-50 microspin columns, GE Healthcare).

After SHAPE and RNP-MaP RNA modification and purification, MaP cDNA synthesis was performed using a revised protocol as described (33 (link)). Briefly, 7 μL of purified modified RNA was mixed with 200 ng of random 9-mer primers and 20 nmol of dNTPs and incubated at 65°C for 10 min followed by 4°C for 2 min. 9 μL 2.22× MaP buffer [1× MaP buffer consists of 6 mM MnCl₂, 1 M betaine, 50 mM Tris (pH 8.0), 75 mM KCl, 10 mM DTT] was added and the combined solution was incubated at 23°C for 2 min. 1 μL SuperScript II Reverse Transcriptase (200 units, Invitrogen) was added and the reverse transcription (RT) reaction was performed according to the following temperature program: 25°C for 10 min, 42°C for 90 min, 10×[50°C for 2 min, 42°C for 2 min], 72°C for 10 min. RT cDNA products were then purified (Illustra G-50 microspin columns, GE Healthcare).