Conservation of m5C in rRNA in the kingdom Plantae

Here we use bisulfite conversion of RNA combined with high-throughput IIlumina sequencing (RBS-seq) to identify single-nucleotide resolution of m5C sites in non-coding ribosomal RNAs of all three sub-cellular transcriptomes across six diverse species that included, the single-celled algae Nannochloropsis oculata, the macro algae Caulerpa taxifolia and multi-cellular higher plants Arabidopsis thaliana, Brassica rapa, Triticum durum and Ginkgo biloba. RNA-seq was performed on total RNA for all species except for Arabidopsis in order to generate rRNA reference sequences using the Arabidopsis rRNA sequences (TAIR10) as a guide. RNA Bisulfite sequencing was used to identify candidate m5C sites in tRNAs and rRNAs of diverse plant species. One biological replicate was performed for each RNA-seq library. One biological replicate was performed for each RNA Bisulfite-seq library except for Arabidopsis thaliana, which was performed in biological triplicate. Illumina sequencing was performed on a MiSeq platform.