An Improved Bacterial Single-cell RNA-seq Reveals Biofilm Heterogeneity

doi:10.21203/rs.3.rs-3329601/v3

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An Improved Bacterial Single-cell RNA-seq Reveals Biofilm Heterogeneity

https://doi.org/10.21203/rs.3.rs-3329601/v3

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In contrast to mammalian cells, bacterial cells lack mRNA polyadenylated tails, presenting a hurdle in isolating mRNA amidst the prevalent rRNA during single-cell RNA-seq. This study introduces a novel method, Ribosomal RNA-derived cDNA Depletion (RiboD), seamlessly integrated into the PETRI-seq technique, yielding RiboD-PETRI. This innovative approach offers a cost-effective, equipment-free, and high-throughput solution for bacterial single-cell RNA sequencing (scRNA-seq). By efficiently eliminating rRNA reads and substantially enhancing mRNA detection rates (up to 92%), our method enables precise exploration of bacterial population heterogeneity. Applying RiboD-PETRI to investigate biofilm heterogeneity, distinctive subpopulations marked by unique genes within biofilms were successfully identified. Notably, PdeI, a marker for the cell-surface attachment subpopulation, was observed to elevate cyclic diguanylate (c-di-GMP) levels, promoting persister cell formation. Thus, we address a persistent challenge in bacterial single-cell RNA-seq regarding rRNA abundance, exemplifying the utility of this method in exploring biofilm heterogeneity. Our method effectively tackles a long-standing issue in bacterial scRNA-seq: the overwhelming abundance of rRNA. This advancement significantly enhances our ability to investigate the intricate heterogeneity within biofilms at unprecedented resolution.

Biological sciences/Microbiology/Biofilms

Biological sciences/Microbiology/Bacteria/Bacterial techniques and applications

bacterial scRNA-seq

biofilms

heterogeneity

The authors declare no competing interests.

movieS1.mp4
Movie S1. Time-lapse images of the persister assay using cells with different PdeI-BFP.
TableS5.SequencingInformation.xls
Table S5. Sequencing information of RiboD-PETRI libraries.
TableS6.ThecostofRiboDPETRI.xls
Table S6. The detailed cost breakdown of RiboD-PETRI.
FigureS1.jpg
Figure S1. (A, B) The number of UMIs detected per cell in recovered cells in different samples (≥15 UMIs/cell): (A) PETRI, (B) RiboD-PETRI at the same unsaturated sequencing depth. The cells are ranked from highest to lowest based on the number of detected UMIs, and cells with ≥15 UMIs are selected for plotting. The median number of UMIs is calculated for these selected cells. (C) Scatterplot illustrating the relationship between reads per cell and counts of UMIs per cell detected from exponential phase E. coli data. Each dot represents a cell. (D) Sequencing saturation of data of exponential period E. coli (3h). We extracted 20%, 40%, 60%, 80% and 100% of the data and further tested their saturation using the saturation calculation method of 10x Genomics. (E & F) Sequencing saturation analysis. We took 20%, 40%, 60%, 80% and 100% of the sequencing data for single-cell analysis and counted the number of genes and UMIs for each cell in these data. The cells were then sorted from largest to smallest values, and cells were taken to count the median number of genes (E) and UMIs (F).
FigureS2.jpg
Figure S2. Comprehensive Single-Cell Transcriptomic Analysis of S. aureus and C. crescentus using RiboD-PETRI. Technical Application of RiboD-PETRI in S. aureus (SA) (A-F), cultured for 9 hours in MHB medium at 37 °C (Table S14) and C. crescentus (CC) (G-L), incubated at 37 °C for 3 hours (Table S15). (A, G) The number of UMIs detected per cell in different samples (≥15 UMIs/cell): (A) S. aureus (SA) and (G) C. crescentus (CC). (B, H) Distribution of mRNA UMIs captured per cell in RiboD-PETRI data of (B) S. aureus (SA) and (H) C. crescentus (CC), presented as violin plots showing the upper quartile, median, and lower quartile lines. The cells are ranked from highest to lowest based on the number of UMIs detected. Then, specific numbers of cells (indicated above the panel) are selected for plotting. The median number of UMIs is calculated for these selected cells. (C, I) The number of genes detected per cell in different samples (C) S. aureus and (I) C. crescentus. The cells are ranked from highest to lowest based on the number of genes detected. Then, specific numbers of cells (indicated above the panel) are selected for plotting. The median number of genes is calculated for these selected cells. "SA" denotes S. aureus, and "CC" denotes C. crescentus. (D, J) UMAP visualization of (D) S. aureus and (J) C. crescentus, demonstrating the ability of RiboD-PETRI to distinguish population heterogeneity. (E, K) Normalized and Principal Component Analysis (PCA) performed on screened data of (E) S. aureus and (K) C. crescentus. The resulting scatterplots show heterogeneity among the populations, with each point representing a cell. (F, L) Distribution of UMIs on the UMAP results for (F) S. aureus and (L) C. crescentus. UMAP results reveal heterogeneity among populations, with each point representing a cell and color shading indicating UMI counts.
TableS10.E.coliRNAseqdata.xls
Table S10. Read per gene of E.coli RNA-seq data in Fig 1E
TableS1.Primersusedinthisstudy.xls
Table S1. Primers used in this study
TableS3.rRNAandmRNAexperssionofPETRIseqandRiboDPETRI.xls
Table S3. Data of rRNA and mRNA experssion of PETRI-seq and RiboD-PETRI.
FigureS3.jpg
Figure S3. Evaluation of Transcriptomic Consistency and Batch Effect Analysis in Static Biofilm E. coli Samples (A) Scatterplot demonstrating the relationship between reads per cell and counts of UMIs per cell detected from static biofilm E. coli data. Two replicates of the sample are included. (B) Calculation of the Pearson correlation coefficient (r) of UMI counts per gene between replicate 1 and replicate 2 of static biofilm E. coli. The analysis involved 4,062 out of 4,141 total genes, with a significant correlation (p-value < 0.0001, r = 0.96), indicating good replication between samples. Each dot represents a gene. (C) Before batch effects were removed, UMAP plot based on the original identity of static biofilm E. coli samples (replicate 1 and replicate 2). Each dot represents a cell, with red indicating replicate 1 and green indicating replicate 2. (D) After batch effects were removed using Harmony, UMAP plot based on the original identity of static biofilm E. coli samples (replicate 1 and replicate 2). (E) Principal Component Analysis (PCA) performed on screened data of two replicates of static biofilm E. coli. The resulting scatterplots show heterogeneity among the populations, with each point representing a cell. (F) Distribution of UMIs on the UMAP results for two replicates of static biofilm E. coli. UMAP results reveal heterogeneity among populations, with each point representing a cell and color shading indicating UMI counts.
TableS4.VariousmethodsinrRNAdepletion.xls
Table S4. The comparison of various methods in rRNA depletion method
FigureS4.jpg
Figure S4. Profiling of Marker Genes in exponential phase E. coli culture by RiboD-PETRI. Expression levels of diverse marker genes across distinct clusters in exponential phase E. coli culture, visualized through violin plots. Each individual dot represents a single cell, demonstrating the high-resolution, single-cell nature of the RiboD-PETRI analysis.
FigureS8.jpg
Figure S8. Schematic chart for the structure of E. coli PdeI.
FigureS5.jpg
Figure S5. Marker Genes Identified in stationary phase S. aureus culture by RiboD-PETRI. Expression levels of different marker genes across different clusters in stationary phase S. aureus culture overlaid on the UMAP plot. Marker genes were selected based on a p-value greater than 0.001 and a log₂FC greater than 0.2. Each dot represents a cell and color shading indicating UMI counts.
FigureS7.jpg
Figure S7. Marker Genes Identified in static E. coli biofilms by RiboD-PETRI. Expression levels of different marker genes across different clusters in static E. coli biofilms overlaid on the UMAP plot. Marker genes were selected based on a p-value greater than 0.001 and a log₂FC greater than 3. Each dot represents a cell and color shading indicating UMI counts.
FigureS6.jpg
Figure S6. Marker Genes Identified in exponential phase C. crescentus culture by RiboD-PETRI. Expression levels of different marker genes across different clusters in exponential phase C. crescentus culture overlaid on the UMAP plot. Marker genes were selected based on a p-value greater than 0.001 and a log₂FC greater than 0.2. Each dot represents a cell and color shading indicating UMI counts.

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An Improved Bacterial Single-cell RNA-seq Reveals Biofilm Heterogeneity

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