Imaging rRNA methylation in bacteria by MR-FISH

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DOI http://dx.doi.org/10.1007/978-1-4939-9674-2_7
Reference K.A. Ganzinger, M. Challand, J. Spencer, D. Klenerman and R.T. Ranasinghe, Imaging rRNA methylation in bacteria by MR-FISH, Methods Mol. Biol. 2038, 89-107 (2019)

Methylation of RNA is normally monitored in purified cell lysates using next-generation sequencing, gel electrophoresis, or mass spectrometry as readouts. These bulk methods require the RNA from ~104 to 107 cells to be pooled to generate sufficient material for analysis. Here we describe a method—methylation-sensitive RNA in situ hybridization (MR-FISH)—that assays rRNA methylation in bacteria on a cell-by-cell basis, using methylation-sensitive hybridization probes and fluorescence microscopy. We outline step-by-step protocols for designing probes, in situ hybridization, and analysis of data using freely available code.