Overview of the CIRS-seq method. Cells are harvested and lysed in isotonic buffer, then treated with Proteinase K to unmask protein-bound regions of RNAs. The whole cell population of RNAs in their native deproteinized conformation is probed with either DMS or CMCT to modify unpaired bases. A non-treated control is also produced to allow further mapping of natural RT stops. After modification, the RNAs from the three populations are reverse transcribed, and cDNA is adapter ligated for high-throughput sequencing. Mapping reads to the transcriptome provide information regarding how many RT stops occurred at each position of the analyzed transcripts. The non-treated (NT) signal at each position is then subtracted from the DMS and CMCT signals to obtain the raw reactivity profile at base resolution. After scaling each data point above the 90th percentile to the 90th percentile, reactivity at each position is divided by the 90th percentile (90% Winsorising) to obtain the normalized reactivity.