An improved bacterial single-cell RNA-seq reveals biofilm heterogeneity
- The State Key Laboratory Breeding Base of Basic Science of Stomatology & Key Laboratory of Oral Biomedicine Ministry of Education, School & Hospital of Stomatology, Medical Research Institute, Wuhan University, China
- Frontier Science Center for Immunology and Metabolism, Wuhan University, China
- Translational Medicine Research Center, North Sichuan Medical College, China
- Center for Life Sciences, School of Life Sciences, Yunnan University, China
- Department of Immunology, Hubei Province Key Laboratory of Allergy and Immunology, State Key Laboratory of Virology and Medical Research Institute, Wuhan University School of Basic Medical Sciences, China
Figures
Figure 1 with 1 supplement
Development of RiboD-PETRI and validation of its technical performance in studying population heterogeneity.
(A) Graphic summary of the RiboD-PETRI method illustrating the incorporation of RiboD after cell pooling and lysis in PETRI-seq. The RiboD protocol is represented by the dashed-line box. In this …
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Figure 1—source code 1
Related to Figure 1.
- https://cdn.elifesciences.org/articles/97543/elife-97543-fig1-code1-v1.zip
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Figure 1—source data 1
Related to Figure 1.
- https://cdn.elifesciences.org/articles/97543/elife-97543-fig1-data1-v1.xls
Figure 1—figure supplement 1
Supplementary analysis of exponential phase E. coli sequencing data.
(A, B) The number of unique molecular identifiers (UMIs) detected per cell in recovered cells in different samples (≥15 UMIs/cell): (A) PETRI, (B) RiboD-PETRI at the same unsaturated sequencing …
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Figure 1—figure supplement 1—source code 1
Related to Figure 1—figure supplement 1.
- https://cdn.elifesciences.org/articles/97543/elife-97543-fig1-figsupp1-code1-v1.zip
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Figure 1—figure supplement 1—source data 1
Related to Figure 1—figure supplement 1.
- https://cdn.elifesciences.org/articles/97543/elife-97543-fig1-figsupp1-data1-v1.xls
Figure 2 with 4 supplements
Comprehensive analysis of single-cell mRNA transcriptomic profiles in exponential phase E. coli using RiboD-PETRI.
(A) The number of unique molecular identifiers (UMIs) detected per cell in recovered cells in exponential period E. coli (≥15 UMIs/cell). The cells are ranked from highest to lowest based on the …
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Figure 2—source code 1
Source code for Figure 2 and Figure 2—figure supplement 2.
- https://cdn.elifesciences.org/articles/97543/elife-97543-fig2-code1-v1.zip
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Figure 2—source code 2
Source code for Figure 2—figure supplement 1, Figure 2—figure supplement 3 and Figure 2—figure supplement 4.
- https://cdn.elifesciences.org/articles/97543/elife-97543-fig2-code2-v1.zip
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Figure 2—source data 1
Related to Figure 2.
- https://cdn.elifesciences.org/articles/97543/elife-97543-fig2-data1-v1.xls
Figure 2—figure supplement 1
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 hr in Mueller-Hinton Broth (MHB) medium at 37°C (Supplementary file 14) and C. crescentus (CC) (G–L), incubated at 37°C …
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Figure 2—figure supplement 1—source data 1
Related to Figure 2—figure supplement 1.
- https://cdn.elifesciences.org/articles/97543/elife-97543-fig2-figsupp1-data1-v1.xls
Figure 2—figure supplement 2
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 …
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Figure 2—figure supplement 2—source code 1
Related to Figure 2—figure supplement 2.
- https://cdn.elifesciences.org/articles/97543/elife-97543-fig2-figsupp2-code1-v1.zip
Figure 2—figure supplement 3
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 Uniform Manifold Approximation and Projection (UMAP) plot. Marker genes …
Figure 2—figure supplement 4
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 Uniform Manifold Approximation and Projection (UMAP) plot. Marker …
Figure 3 with 2 supplements
Single-cell transcriptomic analysis and characterization of static E. coli biofilm using RiboD-PETRI.
(A–F, H) RiboD-PETRI data from static E. coli biofilm (E. coli 24 hr static culture) (Supplementary files 12 and 13). RiboD-PETRI data of static E. coli biofilm were screened for cells with unique …
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Figure 3—source code 1
Source code for Figure 3, Figure 3—figure supplement 1 and Figure 3—figure supplement 2.
- https://cdn.elifesciences.org/articles/97543/elife-97543-fig3-code1-v1.zip
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Figure 3—source data 1
Related to Figure 3.
- https://cdn.elifesciences.org/articles/97543/elife-97543-fig3-data1-v1.xls
Figure 3—figure supplement 1
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 unique molecular identifiers (UMIs) per cell detected from static biofilm E. coli data. Two replicates of the …
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Figure 3—figure supplement 1—source data 1
Related to Figure 3—figure supplement 1.
- https://cdn.elifesciences.org/articles/97543/elife-97543-fig3-figsupp1-data1-v1.xls
Figure 3—figure supplement 2
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 Uniform Manifold Approximation and Projection (UMAP) plot. Marker genes were selected …
Figure 4 with 1 supplement
Functional investigation of marker gene pdeI in static E. coli biofilm.
(A, B) Uniform Manifold Approximation and Projection (UMAP) plots showing the distribution of pdeI in single-cell data of exponential period E. coli (A) and static E. coli biofilm (B). Each dot …
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Figure 4—source data 1
Related to Figure 4.
- https://cdn.elifesciences.org/articles/97543/elife-97543-fig4-data1-v1.xls
Figure 4—figure supplement 1
Schematic chart for the structure of E. coli PdeI.
Author response image 1
The proportion of persister cells in the partially maker genes and empty vector control groups.
Following induction of expression with 0.002% arabinose for 2 hours, a persister counting assay was conducted on the strains using 150 μg/ml ampicillin.
Author response image 2
At almost the same sequencing saturation (64% and 67%), the number of cells exceeding the screening criteria (≥15 UMIs) and the median number of UMIs in cells in Ribod-PETRI and PETRI-seq data of exponential period E. coli (3h).
Videos
Video 1
Time-lapse images of the persister assay using cells with different PdeI-BFP.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Escherichia coli) | MG1655 | Yale Genetic Stock Center | CGSC#6300 | |
| Strain, strain background (Caulobacter crescentus) | NA1000 | Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences | NCBI accession number CP001340 | |
| Strain, strain background (Staphylococcus aureus) | ATCC 25923 | ATCC | ATCC 25923 | |
| Strain, strain background (Escherichia coli) | MG1655 pBAD::gfp | This paper | Figure legends and Materials and methods section | |
| Strain, strain background (Escherichia coli) | MG1655 p(pdeI promoter)::pdeI-gfp | This paper | Figure legends and Materials and methods section | |
| Strain, strain background (Escherichia coli) | MG1655 p(pdeI promoter)::pdeI-bfp | This paper | Figure legends and Materials and methods section | |
| Strain, strain background (Escherichia coli) | MG1655 Δara pBAD::pdeI | This paper | Figure legends and Materials and methods section | |
| Strain, strain background (Escherichia coli) | MG1655 Δara pBAD::vector | This paper | Figure legends and Materials and methods section | |
| Strain, strain background (Escherichia coli) | MG1655 p(pdeI promoter)::bfp | This paper | Figure legends and Materials and methods section | |
| Strain, strain background (Escherichia coli) | MG1655 p(pdeI promoter)::pdeI(G412S)-bfp | This paper | Figure legends and Materials and methods section | |
| Strain, strain background (Escherichia coli) | MG1655 p(pdeI promoter)::bfp p15A::c-di-GMP-sensor | This paper | Figure legends and Materials and methods section | |
| Strain, strain background (Escherichia coli) | MG1655 p(pdeI promoter)::pdeI-bfp p15A::c-di-GMP-sensor | This paper | Figure legends and Materials and methods section | |
| Strain, strain background (Escherichia coli) | MG1655 p(pdeI promoter)::pdeI(G412S)-bfp p15A::c-di-GMP-sensor | This paper | Figure legends and Materials and methods section | |
| Strain, strain background (Escherichia coli) | MG1655 Δara pBAD::bfp | This paper | Figure legends and Materials and methods section | |
| Recombinant DNA reagent | p15A::c-di-GMP-sensor | This paper | p15A ori | |
| Recombinant DNA reagent | pBAD::vector | This paper | Arabinose-induction | |
| Recombinant DNA reagent | pBAD::gfp | This paper | Arabinose-induction | |
| Recombinant DNA reagent | pBAD::bfp | This paper | Arabinose-induction | |
| Recombinant DNA reagent | p(pdeI promoter)::bfp | This paper | pdeI native promoter induction | |
| Recombinant DNA reagent | p(pdeI promoter)::pdeI-bfp | This paper | pdeI native promoter induction | |
| Recombinant DNA reagent | p(pdeI promoter)::pdeI(G412S)-bfp | This paper | pdeI native promoter induction | |
| Recombinant DNA reagent | p(pdeI promoter)::pdeI-gfp | This paper | pdeI native promoter induction | |
| Recombinant DNA reagent | p(pdeI promoter)::pdeI | This paper | pdeI native promoter induction | |
| Recombinant DNA reagent | pBAD::pdeI-gfp | This paper | Arabinose-induction | |
| Sequence-based reagent | P-pdeI-F | This paper | PCR primers | AATTGTCTGATTCGTTACCAACTGACCGTACTGGCGTTC |
| Sequence-based reagent | P-pdeI-R | This paper | PCR primers | TTGCTGCTGCCTCGGCTTCTAGCTCTTTTACTAATTTTCCACTTTTATCCCAGG |
| Sequence-based reagent | pdeI-F | This paper | PCR primers | GGCTAACAGGAGGAATTAACCATGCTGAGTTTATACGAAAAGATAAAGATAAG |
| Sequence-based reagent | pdeI-R | This paper | PCR primers | GCTGGAGACCGTTTAAACTCACTACTCTTTTACTAATTTTCCACTTTTATCCC |
| Sequence-based reagent | pBAD-R | This paper | PCR primers | TTGGTAACGAATCAGACAATTGAC |
| Sequence-based reagent | pBAD-F | This paper | PCR primers | TGAGTTTAAACGGTCTCCAGC |
| Sequence-based reagent | pBAD-R2 | This paper | PCR primers | GGTTAATTCCTCCTGTTAGCCC |
| Sequence-based reagent | Bfp-F | This paper | PCR primers | CGAGGCAGCAGCAAAGGCCCTAGAAGGTGGATCCGGCGGTTCTAG |
| Sequence-based reagent | Gfp-F | This paper | PCR primers | CTAGAAGCCGAGGCAGCAGCAAAGGCCCTAGAAATGAGTAAAGGAGAAGAACTTTTCAC |
| Sequence-based reagent | G412S-F | This paper | PCR primers | GAAGCGGTGTTTAGTGTTGATG |
| Sequence-based reagent | G412S-R | This paper | PCR primers | CATCAACACTAAACACCGCTTC |
| Sequence-based reagent | P-bfp-R | This paper | PCR primers | GTTAATACATTTAACAAAATAACTATCTGA |
| Sequence-based reagent | P-bfp-F | This paper | PCR primers | ATAGTTATTTTGTTAAATGTATTAACGGTGGATCCGGCGGTTCT |
| Sequence-based reagent | UP-F | This paper | PCR primers | CATGAATTCTGGCGACGATTTCG |
| Sequence-based reagent | UP-R | This paper | PCR primers | GTTAATACATTTAACAAAATAACTATCTGA |
| Sequence-based reagent | ccdB-F | This paper | PCR primers | CACAGCGTTCAGATAGTTATTTTGTTAAATGTATTAACTCTAGAGCGACGCCAGACG |
| Sequence-based reagent | ccdB-R | This paper | PCR primers | CTGTAAGTACGAACTTATTGATTCTGGACATACGTAAATTACGCCCCGCCCTGCCAC |
| Sequence-based reagent | Down-F | This paper | PCR primers | TTTACGTATGTCCAGAATCAATAAGTTCGTACTTAC |
| Sequence-based reagent | Down-R | This paper | PCR primers | ATCTTCGTCAAAGGATTTTCTGCCC |
| Sequence-based reagent | UP2-R | This paper | PCR primers | ATCTTTTCGTATAAACTCAGCATGTTAATACATTTAACAAAATAACTATCTGAA |
| Sequence-based reagent | pdeI-G412S-F | This paper | PCR primers | ATGCTGAGTTTATACGAAAAGATAAAGAT |
| Sequence-based reagent | pdeI-G412S-R | This paper | PCR primers | CTTATTGATTCTGGACATACGTAAACTACTCTTTTACTAATTTTCCACT |
| Sequence-based reagent | Down2-F | This paper | PCR primers | TTTACGTATGTCCAGAATCAATAAGTTCGTACTTAC |
| Commercial assay or kit | KAPA HIFI hotStart ReadyMix PCR Kits | KAPA | Cat#2602 | |
| Commercial assay or kit | VAHTS Universal DNA Library Prep Kit | Vazyme | Cat#NR603 | |
| Commercial assay or kit | Bacteria RNA Extraction Kit | Vazyme | Cat#R403-01 | |
| Commercial assay or kit | Ribo-off rRNA Depletion Kit (Bacteria) | Vazyme | Cat#N407 | |
| Commercial assay or kit | 2× MultiF Seamless Assembly Mix | ABclonal | Cat#RK21020 | |
| Commercial assay or kit | VAHTS Universal DNA Library Prep Kit for Illumina V3 | Vazyme | Cat#ND607 | |
| Commercial assay or kit | ABScript III RT Master Mix for qPCR with gDNA Remover | ABclonal | Cat#RK20429 | |
| Commercial assay or kit | SUPERase-In RNase Inhibitor | Invitrogen | Cat#AM2696 | |
| Chemical compound, drug | Streptavidin Magnetic Beads | Thermo Fisher | Cat#88816 | |
| Chemical compound, drug | Syto 24 dye | Invitrogen | Cat#S7559 | |
| Chemical compound, drug | Arabinose | Sigma | Cat#V900920 | |
| Chemical compound, drug | Ampicillin | Sangon Biotech | Cat#A610028 | |
| Chemical compound, drug | Chloramphenicol | Sangon Biotech | Cat#A600118 | |
| Chemical compound, drug | Kanamycin | Sangon Biotech | Cat#A600286 | |
| Software, algorithm | Fiji | GitHub | https://fiji.sc/; RRID:SCR_002285 | |
| Software, algorithm | FlowJo | Treestar, Inc | https://www.flowjo.com/ |
Additional files
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Supplementary file 1
Primers used in this study.
Related to RiboD-PETRI library construction.
- https://cdn.elifesciences.org/articles/97543/elife-97543-supp1-v1.xls
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Supplementary file 2
Multiplet frequency.
The specific calculation process for multiplet frequency.
- https://cdn.elifesciences.org/articles/97543/elife-97543-supp2-v1.xls
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Supplementary file 3
rRNA and mRNA expression of PETRI-seq and RiboD-PETRI.
- https://cdn.elifesciences.org/articles/97543/elife-97543-supp3-v1.xls
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Supplementary file 4
Various methods in rRNA depletion.
- https://cdn.elifesciences.org/articles/97543/elife-97543-supp4-v1.xls
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Supplementary file 5
Sequencing information.
The detailed information of RiboD-PETRI libraries.
- https://cdn.elifesciences.org/articles/97543/elife-97543-supp5-v1.xls
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Supplementary file 6
The cost of RiboD-PETRI.
The detailed cost breakdown of RiboD-PETRI.
- https://cdn.elifesciences.org/articles/97543/elife-97543-supp6-v1.xls
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Supplementary file 7
Matrix_of_E. coli_3h_data_by_RiboD-PETRI_in_Figure 1C–E.
- https://cdn.elifesciences.org/articles/97543/elife-97543-supp7-v1.zip
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Supplementary file 8
Matrix_of_E. coli_data_by_PETRI-seq_in_Figure 1C.
- https://cdn.elifesciences.org/articles/97543/elife-97543-supp8-v1.zip
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Supplementary file 9
Matrix_of_E. coli_data_by_PETRI-seq_in_Figure 1D.
- https://cdn.elifesciences.org/articles/97543/elife-97543-supp9-v1.zip
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Supplementary file 10
E. coli RNA-seq data.
The result of bulk RNA-seq of exponential period E. coli sample in Figure 1E.
- https://cdn.elifesciences.org/articles/97543/elife-97543-supp10-v1.xls
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Supplementary file 11
Matrix_of_Exponential_period_E. coli_data.
- https://cdn.elifesciences.org/articles/97543/elife-97543-supp11-v1.zip
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Supplementary file 12
Matrix_of_Static_E. coli_biofilm-1_data.
- https://cdn.elifesciences.org/articles/97543/elife-97543-supp12-v1.zip
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Supplementary file 13
Matrix_of_Static_E. coli_biofilm-2_data.
- https://cdn.elifesciences.org/articles/97543/elife-97543-supp13-v1.zip
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Supplementary file 14
Matrix_of_SA_data.
- https://cdn.elifesciences.org/articles/97543/elife-97543-supp14-v1.zip
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Supplementary file 15
Matrix_of_CC_data.
- https://cdn.elifesciences.org/articles/97543/elife-97543-supp15-v1.zip
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MDAR checklist
- https://cdn.elifesciences.org/articles/97543/elife-97543-mdarchecklist1-v1.docx
