Sequencing-Based RNA Structure Analysis

From each sample of RNA (defined above, E. coli or murine total RNA, extracted and SHAPEmodified or untreated), 1-3 μg were subjected to MaP reverse transcription [requiring Superscript II and addition of Mn²⁺ to the RT buffer(25 (link), 39 (link))] using random nonamer primers. The cDNA generated was buffer exchanged (Illustra microspin G-50 columns, GE Healthcare) and the volume increased to 68 μL. For second-strand cDNA synthesis, 8 μL of 10× buffer (Second Strand Synthesis Reaction Buffer, NEB) and 4 μL enzyme (Second Strand Synthesis Enzyme mix, NEB) were added to the cDNA product and incubated for 2.5 h at 16 °C. Double-stranded cDNA was fragmented and amplified with sequencing indexes (Nextera XT library prep kit, Illumina). Nextera PCR products were affinity purified (using a 0.8× ratio of Agencourt AMPure XP beads, Beckman Coulter) and eluted in 20 μL of nuclease-free water.