Presentation Information
[P03-351]Development of large-scale bacterial bulk RNA-seq and its applications to temporal transcriptome analysis
○Mika Nishimura1, Kazuki Takahashi2, Kaori Aikawa2, Tetsutaro Hayashi3, Mariko Kuse3, Itoshi Nikaido3,4, Haruko Takeyama1,2,5, Masahito Hosokawa1,2,5 (1. Grad. Sch. Adv. Sci. Eng., Waseda Univ. (Japan), 2. Res. Org. Nano Life Innov., Waseda Univ. (Japan), 3. RIKEN, TRIP (Japan), 4. Med. Res. Inst., Science Tokyo (Japan), 5. Inst. Adv. Res. Biosyst. Dyn., Waseda Res. Inst. Sci. Eng., Waseda Univ. (Japan))
Keywords:
Bacteria,Transcriptome,RNA-seq,High-throughput
[Purpose]
RNA-seq has rapidly advanced since the 2000s as a comprehensive gene expression analysis method. While its primary focus was eukaryotes, the scope of target organisms has gradually expanded, and the technology is increasingly being applied to bacterial populations. Bacteria have been utilized across a wide range of industrial fields by leveraging their high material production capability and have recently seen gained further attention for their role in bio-manufacturing. In bio-manufacturing, comprehensive analysis through multi-omics approaches is effective for optimizing material production capabilities, and RNA-seq plays a part in this. Conventional bulk bacterial RNA-seq involves a workflow consisting of cell wall disruption, RNA extraction and purification on column, reverse transcription, and library preparation. The steps up to RNA extraction involve independent operations for each sample, resulting in low throughput and conventional techniques have limitations in scalability. Therefore, this research has focused on developing methods aimed at scaling up bacterial bulk RNA-seq technology.
[Method]
(i) Method Development
The single-cell RNA sequencing (scRNA-seq) technology, which acquires gene expression for each individual cell, excels in parallel processing. Therefore, this study aimed to scale up a bacterial bulk RNA-seq technology based on scRNA-seq technology. Building upon our previously reported plate-based bacterial scRNA-seq technology1), we established a large-scale bacterial bulk RNA-seq. This method uses bacterial culture equivalent to 1000 cells as input and employs a series of continuous enzymatic reactions on plates.
(ii) Performance Evaluation
The developed method was evaluated using E. coli, comparing with conventional bulk RNA-seq for detection power, accuracy, and cost.
(iii) Applied Analysis
As an analysis example using our method, we performed time-course analysis during the growth and multi-condition comparative analysis of drug responses.
[Results]
The development method demonstrated high throughput, enabling processing of approximately four 96-well plates per day up to the second-strand synthesis reaction. Furthermore, it could acquire gene expression patterns showing high correlation (R ≒ 0.7) with conventional techniques at a highly cost-effective rate (approximately 9 times cheaper). Enabling large-scale analysis, it captured short-term gene expression dynamics with high temporal resolution at 15-minute intervals and realized multi-condition comparisons by setting multiple conditions such as strain, drug, and time point.
[Conclusion]
In summary, this study achieved the large-scale bacterial bulk RNA-seq. By enabling extensive gene expression analysis across diverse bacterial species and cultivation conditions, it is expected to contribute not only to bio-manufacturing but also to deepening physiological understanding of bacterial populations.
1) Nishimura, M. et al., J. Biosci. Bioeng. (2023)
RNA-seq has rapidly advanced since the 2000s as a comprehensive gene expression analysis method. While its primary focus was eukaryotes, the scope of target organisms has gradually expanded, and the technology is increasingly being applied to bacterial populations. Bacteria have been utilized across a wide range of industrial fields by leveraging their high material production capability and have recently seen gained further attention for their role in bio-manufacturing. In bio-manufacturing, comprehensive analysis through multi-omics approaches is effective for optimizing material production capabilities, and RNA-seq plays a part in this. Conventional bulk bacterial RNA-seq involves a workflow consisting of cell wall disruption, RNA extraction and purification on column, reverse transcription, and library preparation. The steps up to RNA extraction involve independent operations for each sample, resulting in low throughput and conventional techniques have limitations in scalability. Therefore, this research has focused on developing methods aimed at scaling up bacterial bulk RNA-seq technology.
[Method]
(i) Method Development
The single-cell RNA sequencing (scRNA-seq) technology, which acquires gene expression for each individual cell, excels in parallel processing. Therefore, this study aimed to scale up a bacterial bulk RNA-seq technology based on scRNA-seq technology. Building upon our previously reported plate-based bacterial scRNA-seq technology1), we established a large-scale bacterial bulk RNA-seq. This method uses bacterial culture equivalent to 1000 cells as input and employs a series of continuous enzymatic reactions on plates.
(ii) Performance Evaluation
The developed method was evaluated using E. coli, comparing with conventional bulk RNA-seq for detection power, accuracy, and cost.
(iii) Applied Analysis
As an analysis example using our method, we performed time-course analysis during the growth and multi-condition comparative analysis of drug responses.
[Results]
The development method demonstrated high throughput, enabling processing of approximately four 96-well plates per day up to the second-strand synthesis reaction. Furthermore, it could acquire gene expression patterns showing high correlation (R ≒ 0.7) with conventional techniques at a highly cost-effective rate (approximately 9 times cheaper). Enabling large-scale analysis, it captured short-term gene expression dynamics with high temporal resolution at 15-minute intervals and realized multi-condition comparisons by setting multiple conditions such as strain, drug, and time point.
[Conclusion]
In summary, this study achieved the large-scale bacterial bulk RNA-seq. By enabling extensive gene expression analysis across diverse bacterial species and cultivation conditions, it is expected to contribute not only to bio-manufacturing but also to deepening physiological understanding of bacterial populations.
1) Nishimura, M. et al., J. Biosci. Bioeng. (2023)
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