Deep sequencing data for document titled: Rolling circle RNA synthesis catalysed by RNA

RNA-catalysed RNA replication is widely considered a key step in the emergence of life’s first genetic system. However, RNA replication can be impeded by the extraordinary stability of duplex RNA products, which must be dissociated for re-initiation of the next replication cycle. Here we have explored rolling circle synthesis (RCS) as a potential solution to this strand separation problem. RCS on small circular RNAs - as indicated by molecular dynamics simulations - induces a progressive build-up of conformational strain with destabilisation of nascent strand 5’ and 3’ ends. At the same time, we observe sustained RCS by a triplet polymerase ribozyme on small circular RNAs over multiple orbits with strand displacement yielding concatemeric RNA products. Furthermore, we show RCS of a circular Hammerhead ribozyme capable of self-cleavage and re-circularisation. Thus, all steps of a viroid-like RNA replication pathway can be catalysed by RNA alone. Our results have implications for the emergence of RNA replication and for understanding the potential of RNA to support complex genetic processes.

Methods
RNA from primer extension reactions catalysed by the triplet polymerase ribozyme were PAGE purified, adaptor ligated and prepared for illumina sequencing as described in the main text.

Illumina Sequencing data were acquired and processed as FASTQ files using Terminal (and available software packages such as FASTX-toolkit). Prior to analysis the whole output file form illumina sequencing runs (containing also unrelated sequences) was split based on barcodes identifying the individual samples and trimmed starting with the original (P91) primer sequence (GAAGAACTG). After the primer sequence, the triplets at positions 1, 2 ,3 etc. would be identifiable representing extension products made by the ribozyme. The presence for the 3’ adapter sequence (GTCGAATAT…) in the aligned sequences marked the end of the original RNA extension product.

File 1 include the split and trimmed sequence data for circular and linear one-pot analysis (Circular_onepot and Linear_onepot, respectively), File 2 include the split and trimmed sequence data for branched RCS analysis (B3)).