Extended Data Fig. 2: Piloting single-cell RNA-sequencing in bacteria without rRNA depletion. | Nature Microbiology

Extended Data Fig. 2: Piloting single-cell RNA-sequencing in bacteria without rRNA depletion.

From: Single-cell massively-parallel multiplexed microbial sequencing (M3-seq) identifies rare bacterial populations and profiles phage infection

Extended Data Fig. 2

a. Distributions represent bacterial cells per droplet produced on the Chromium Controller (10x Genomics) at indicated cell loading numbers. Bacterial droplet loading was quantified as described in Materials and Methods. b. Representative image of droplets quantified in panel (A). E. coli cells, which were stained with Sytox green, are visible as green dots. c. Curves represent expected index collision rates (that is, percentage of cells with the same round-one index labelled with the same round-two index) as a function of loaded cells using different indexing schemes. d. Analysis of a mixture of exponential and stationary phase B. subtilis (blue) and E. coli (red) using only round-two (droplet-based) indexes. Species assignments for each ‘cell’ were made as determined in Materials and Methods. Data were generated without rRNA depletion (eBW1 in Supplementary Table 2). e. Same as (D) but analysis performed with combinatorial barcodes. f. Comparison of bulk RNA-seq data to pseudobulk, computationally rRNA-depleted single-cell gene expression in exponential phase E. coli from eBW1. Each point represents a single gene. r, Pearson correlation. g. Read counts per cell from single-cell gene expression data without rRNA depletion (1023 ± 436 reads for B. subtilis and 1926 ± 1054 reads for E. coli when considering rRNAs; 117 ± 81 reads for B. subtilis and 72 ± 72 reads for E. coli when considering only non-rRNAs). Data was collected from a single experiment; over B. subtilis 4601 cells and E. coli 5883 cells. Boxplot limits are as defined in Materials and Methods. h. Same as (G) but for UMI counts per cell (34 ± 13 UMIs for B. subtilis and 52 ± 23 UMIs for E. coli when considering rRNAs; 5 ± 5 UMIs for B. subtilis and 3 ± 3 UMIs for E. coli when considering only non-rRNAs). i. Same as (G) but for stationary phase B. subtilis (1838 cells) and E. coli (2094 cells) (720 ± 380 reads for B. subtilis and 1902 ± 1297 reads for E. coli when considering rRNAs; 23 ± 23 reads for B. subtilis and 0 ± 0 reads for E. coli when considering only non-rRNAs). j. Same as (H) but for stationary phase B. subtilis and E. coli (22 ± 10 UMIs for B. subtilis and 50 ± 30 UMIs for E. coli when considering rRNAs; 2 ± 2 UMIs for B. subtilis, and 0 ± 0 UMIs for E. coli when considering only non-rRNAs). Data above are reported as medians with maximum average deviation.

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