Lunch Pitches with Joram Waititu, Aicha Kriaa, Anna Morén and Marianne Eik

Bacterial populations exhibit significant heterogeneity, allowing distinct cell subgroups to thrive in diverse environmental stress. Recent advancements in single-cell techniques, particularly single-cell RNA sequencing (scRNA-seq), offer a powerful means to unravel cellular heterogeneity during environmental stress, infections and discern subpopulation variations that influence individual cell outcomes. However, current bacterial scRNA-seq methods suffer from the low number of cells being sequenced and the low number of transcripts detected in each cell due to leakage of nucleic acids due to mechanical disruption of cell walls during permeabilization for subsequent enzymatic reactions. Here, I will highlight a novel microfluidic bacterial scRNA-seq method we are developing by using the advantage of semi-permeable hydrogel microcapsules (SPCs). Due to the selective permeability of the SPCs, the desirable enzymes and reagents can be loaded, or replaced, in the microcapsule at any given step by simply changing the reaction buffer in which the SPCs are dispersed. Therefore, complex molecular biology workflows can be readily adapted to conduct nucleic acid analysis on encapsulated mammalian cells, or other biological species. The SPCs support sequential multi-step enzymatic reactions and remain intact under different biochemical conditions, such as freezing, thawing, and thermocycling. This comprehensive approach overcomes technical obstacles associated with sequencing bacterial cells, enabling the sequencing over a hundred thousand cells. This method will provide valuable insights that offer a deeper understanding of the molecular mechanisms underlying bacterial heterogeneity during infection, thus opening advancements in our understanding of bacterial pathogenesis.