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Q Are there restrictions on the shape and size of bacteria?A
There are no restrictions regarding the shape or size of bacteria. The typical diameter of bacteria ranges from 0.5-10 µm, which meets the detection criteria.
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Q What defines a "single species" in the context of the VITA MscRNA-seq High-Throughput Single-Species Single-Bacterium Transcriptome? Does it refer to a single strain, or does it encompass multiple strains of the same species?A
In this context, "single species" pertains to the taxonomic level of the bacterial species, rather than individual strains within the species.
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Q Is enrichment culturing requried for preparing single-bacterium suspensions from gut microbiota samples for single-bacterium RNA sequencing?A
No, enrichment culturing is not required. However, sample pretreatment, including washing with PBST containing RNase inhibitor, mixing, filtration, and centrifugation, is necessary to remove impurities and efficiently collect bacterial cells.
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Q What are the recommended number of bacterial cells to capture and the suggested sequencing depth?A
For experiments involving a single bacterial species, we recommend capturing 2,000-3,000 cells per sample with a sequencing depth of 100-200 million reads.
For experiments with mixed bacterial species, it is advisable to capture 5,000-10,000 cells per sample with a sequencing depth of 500-650 million reads.
However, the VITA single-cell platform allows for the capture of up to 20,000 cells per sample, providing the flexibility to adjust bacterial cell counts based on specific experimental objectives and needs.
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Q How can I address the issue of high ribosomal RNA content?A
To overcome the challenge of high ribosomal RNA (rRNA) content in bacterial cells, increasing sequencing depth is essential. This strategy ensures the detection of low-abundance RNA molecules, facilitating robust clustering and accurate gene expression analysis.
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Q What are the advantages of single-bacterium RNA sequencing compared to bulk-RNA seq of bacteria?A
Single-bacterium RNA sequencing can reveal the heterogeneity among individual bacterial cells within a population, a detail that is obscured in bulk RNA sequencing where gene expression is averaged across the sample. This capability allows for precise investigations into how individual bacterial cells respond to various environmental pressures or treatments. It is also essential for uncovering complex phenomena such as heteroresistance and analyzing functional dynamics within mixed bacterial populations.
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Q Can the VITA platform detect differences in transcription levels between wild-type strains and mutant strains created through point mutation modification?A
Yes, as long as there are significant transcriptional differences between the mutant and wild-type strains, the VITA platform can detect them.
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Q Can VITA prokaryotic single-cell RNA sequencing data accurately reflect the abundance of different bacterial species within the microbiota? How consistent are these results compared to those obtained from metatranscriptomics?A
Yes, VITA prokaryotic single-cell RNA sequencing data can accurately reflect the abundance of various bacterial species, with minor differences compared to metatranscriptomics. The predominant species detected by both methods generally show a high degree of consistency.
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Q Does the functional analysis of single-bacteria transcriptomes require reference genomes?A
Yes, reference genomes are required.
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Q Can VITA prokaryotic single-cell RNA sequencing be used to investigate interactions between different bacterial populations/subpopulations?A
Yes, the VITA platform supports the analysis of interactions among multiple bacterial populations/subpopulations.
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Q After identifying bacterial subpopulations with specific functions using the VITA platform, is further functional validation necessary?A
The requirement for functional validation depends on your research objectives. If validating the bacterial subpopulations identified by the VITA platform is crucial for your study, methods such as constructing fluorescent reporter strains or employing flow cytometry sorting may be utilized.
