Abstract: In one example embodiment, methods of generating a single prokaryotic cell cDNA library include barcoding RNAs from different prokaryotic cells individually, allowing cell identity of each RNA to be retained in a single sequencing library. In one example embodiment, methods include, prior to generating a cDNA library, degrading rRNA and DNA in each cell of a set of fixed and permeabilized cells. In one example embodiment, methods include converting mRNA in each fixed and permeabilized cell into cDNA labeled with a cell-specific first and second barcode combination and a unique molecular identifier (UMI). In one example embodiment, methods include amplifying labeled cDNA and preparing a sequencing library of amplified labeled cDNA.