Extended Data Fig. 1: M3-seq experimental workflow and rRNA depletion scheme.

a. Detailed schematic of M3-seq experimental workflow: Populations of fixed and permeabilized bacteria are (i) aliquoted into wells of one or more 96 well plates. Each well contains a uniquely indexed random hexamer, which acts as a primer for (ii) in situ reverse transcription. These primers also carry unique molecular identifier (UMIs) sequences. During reverse transcription, cell-associated RNA molecules are converted to cDNAs with primer barcodes and UMIs on their 5’ ends. After reverse transcription, (iii) cells are pooled and loaded into a commercially available device for droplet-based indexing (herein, the Chromium Controller from 10x Genomics) without a need for limiting dilution. After partitioning into droplets, (iv) a second index is ligated onto the 5’ end of the reverse transcribed, cell-associated cDNAs (herein, using Next GEM Single Cell ATAC reagents from 10x Genomics). Following indexing, cells are lysed, and (v) cDNA molecules are converted to double-strand DNA using a Klenow enzyme and a random primer with a PCR handle at the 5’ end. This double-strand cDNA is then (vi) amplified by PCR, (vii) fragmented with Tn5 transposase loaded with Nextera read 2 primers, and (viii) attached to a T7 promoter via a second round of PCR. Next, (ix) cDNA molecules are transcribed back into RNA using T7 RNA polymerase. This step prepares the amplified library for rRNA depletion. After transcription, (x) the resulting RNA is annealed to a set of DNA probes that are complementary to rRNA sequences within the library (Supplementary Table 4). This annealing allows for selective degradation of those sequences with RNase H. Finally, in a second reverse transcription step, (xi) the indexed and rRNA-depleted library is converted back into cDNA, and (xii) the resulting cDNA is amplified one more time to add a required sequencing adaptor. The library is then ready for paired-end sequencing. b. Detailed schematic of rRNA depletion steps: To remove rRNA sequences from M3-seq libraries, we (i) convert indexed and amplified cDNA libraries into RNA via in vitro transcription, (ii) hybridized rRNA sequences within the library to DNA probes and digest those sequences using RNase H, and (iii) convert the remaining sequences back into DNA using a P5 primer.