Fig. 1: Developing a sequencing-based approach to detect Tb³⁺ cleavage sites.

a 3D structure of group II intron with insert showing localization of metal ion in region of negative electrostatic potential region Adapted from PDB “4E8M”. b Mechanism of Tb³⁺ mediated cleavage. c Denaturing electrophoresis of ³²P-labeled aI5γ RNA probed at the indicated TbCl₃ concentrations. Source data are provided as a Source Data file. N = 2. d Primer extension electrophoresis of corresponding cDNA products from reverse transcription. Source data are provided as a Source Data file. N = 2. e Tb-seq library preparation workflow. A Tb³⁺ cleaved RNA and an untreated RNA are reverse transcribed with MarathonRT and a gene-specific RT primer containing a 5’ adapter handle. Illumina adapter (purple). The resulting cDNAs are ligated to a random hexamer fused to an Illumina adapter (pink) and PCR amplified to incorporate Illumina multiplex handles. Stop sites are processed using the RTEvents counter script (see Methods).