The field of transcriptomics examines cellular RNA content and provides functional insight into organisms, their regulatory mechanisms and responses to changing environmental conditions. Initial methods for transcriptomic analysis required high amount of input material and provided an averaged-out, population level readout. However, biological systems are inherently heterogenous. To probe this heterogeneity, single-cell transcriptomic technology was developed [1]. The technology became inseparable in the fields of cancer and developmental biology among many others, however, its’ application in the prokaryotic research remained relatively constrained [2].This is due several limitations: most prokaryotes possess a rigid cell wall, complicating the cell lysis, the amount of bacterial messenger RNA (mRNA) is low compared to eukaryotes, molecules have a shorter lifespan, usually lasting only few minutes, and lack a poly-A tail, required for barcoding. Cellular fixation overcomes some of these hurdles, however, to enable efficient transcriptomic studies, bacterial RNA must be successfully barcoded, reverse transcribed and amplified. In this work, fixation reagents: formaldehyde, glutaraldehyde and paraformaldehyde (PFA) were evaluated and optimal concentrations and fixation durations determined. What is more, several approaches to reverse transcribe bacterial mRNA were explored, including polyadenylation prior reverse transcription (RT) versus the use of random hexamer primers.
To this end, PFA fixed bacterial samples underwent isolation in semi-permeable microcapsules, lysis, polyadenylation, reverse transcription, PCR and fluorescent imaging.