Efficient and cost-effective bacterial mRNA sequencing from low input samples through ribosomal RNA depletion
Abstract Background RNA sequencing is a powerful approach to quantify the genome-wide distribution of mRNA molecules in a population to gain deeper understanding of cellular functions and phenotypes. However, unlike eukaryotic cells, mRNA sequencing of bacterial samples is more challenging due to th...
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| Format: | Article |
| Language: | English |
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BMC
2020-10-01
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| Series: | BMC Genomics |
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| Online Access: | http://link.springer.com/article/10.1186/s12864-020-07134-4 |
| _version_ | 1819194832614064128 |
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| author | Chatarin Wangsanuwat Kellie A. Heom Estella Liu Michelle A. O’Malley Siddharth S. Dey |
| author_facet | Chatarin Wangsanuwat Kellie A. Heom Estella Liu Michelle A. O’Malley Siddharth S. Dey |
| author_sort | Chatarin Wangsanuwat |
| collection | DOAJ |
| description | Abstract Background RNA sequencing is a powerful approach to quantify the genome-wide distribution of mRNA molecules in a population to gain deeper understanding of cellular functions and phenotypes. However, unlike eukaryotic cells, mRNA sequencing of bacterial samples is more challenging due to the absence of a poly-A tail that typically enables efficient capture and enrichment of mRNA from the abundant rRNA molecules in a cell. Moreover, bacterial cells frequently contain 100-fold lower quantities of RNA compared to mammalian cells, which further complicates mRNA sequencing from non-cultivable and non-model bacterial species. To overcome these limitations, we report EMBR-seq (Enrichment of mRNA by Blocked rRNA), a method that efficiently depletes 5S, 16S and 23S rRNA using blocking primers to prevent their amplification. Results EMBR-seq results in 90% of the sequenced RNA molecules from an E. coli culture deriving from mRNA. We demonstrate that this increased efficiency provides a deeper view of the transcriptome without introducing technical amplification-induced biases. Moreover, compared to recent methods that employ a large array of oligonucleotides to deplete rRNA, EMBR-seq uses a single or a few oligonucleotides per rRNA, thereby making this new technology significantly more cost-effective, especially when applied to varied bacterial species. Finally, compared to existing commercial kits for bacterial rRNA depletion, we show that EMBR-seq can be used to successfully quantify the transcriptome from more than 500-fold lower starting total RNA. Conclusions EMBR-seq provides an efficient and cost-effective approach to quantify global gene expression profiles from low input bacterial samples. |
| first_indexed | 2024-12-23T02:03:08Z |
| format | Article |
| id | doaj.art-406a211968574b98b582088fbc8c6a84 |
| institution | Directory Open Access Journal |
| issn | 1471-2164 |
| language | English |
| last_indexed | 2024-12-23T02:03:08Z |
| publishDate | 2020-10-01 |
| publisher | BMC |
| record_format | Article |
| series | BMC Genomics |
| spelling | doaj.art-406a211968574b98b582088fbc8c6a842022-12-21T18:03:56ZengBMCBMC Genomics1471-21642020-10-0121111210.1186/s12864-020-07134-4Efficient and cost-effective bacterial mRNA sequencing from low input samples through ribosomal RNA depletionChatarin Wangsanuwat0Kellie A. Heom1Estella Liu2Michelle A. O’Malley3Siddharth S. Dey4Department of Chemical Engineering, University of California Santa BarbaraDepartment of Chemical Engineering, University of California Santa BarbaraDepartment of Chemical Engineering, University of California Santa BarbaraDepartment of Chemical Engineering, University of California Santa BarbaraDepartment of Chemical Engineering, University of California Santa BarbaraAbstract Background RNA sequencing is a powerful approach to quantify the genome-wide distribution of mRNA molecules in a population to gain deeper understanding of cellular functions and phenotypes. However, unlike eukaryotic cells, mRNA sequencing of bacterial samples is more challenging due to the absence of a poly-A tail that typically enables efficient capture and enrichment of mRNA from the abundant rRNA molecules in a cell. Moreover, bacterial cells frequently contain 100-fold lower quantities of RNA compared to mammalian cells, which further complicates mRNA sequencing from non-cultivable and non-model bacterial species. To overcome these limitations, we report EMBR-seq (Enrichment of mRNA by Blocked rRNA), a method that efficiently depletes 5S, 16S and 23S rRNA using blocking primers to prevent their amplification. Results EMBR-seq results in 90% of the sequenced RNA molecules from an E. coli culture deriving from mRNA. We demonstrate that this increased efficiency provides a deeper view of the transcriptome without introducing technical amplification-induced biases. Moreover, compared to recent methods that employ a large array of oligonucleotides to deplete rRNA, EMBR-seq uses a single or a few oligonucleotides per rRNA, thereby making this new technology significantly more cost-effective, especially when applied to varied bacterial species. Finally, compared to existing commercial kits for bacterial rRNA depletion, we show that EMBR-seq can be used to successfully quantify the transcriptome from more than 500-fold lower starting total RNA. Conclusions EMBR-seq provides an efficient and cost-effective approach to quantify global gene expression profiles from low input bacterial samples.http://link.springer.com/article/10.1186/s12864-020-07134-4Bacterial mRNA sequencingmRNA enrichmentrRNA depletionLow input total RNA |
| spellingShingle | Chatarin Wangsanuwat Kellie A. Heom Estella Liu Michelle A. O’Malley Siddharth S. Dey Efficient and cost-effective bacterial mRNA sequencing from low input samples through ribosomal RNA depletion BMC Genomics Bacterial mRNA sequencing mRNA enrichment rRNA depletion Low input total RNA |
| title | Efficient and cost-effective bacterial mRNA sequencing from low input samples through ribosomal RNA depletion |
| title_full | Efficient and cost-effective bacterial mRNA sequencing from low input samples through ribosomal RNA depletion |
| title_fullStr | Efficient and cost-effective bacterial mRNA sequencing from low input samples through ribosomal RNA depletion |
| title_full_unstemmed | Efficient and cost-effective bacterial mRNA sequencing from low input samples through ribosomal RNA depletion |
| title_short | Efficient and cost-effective bacterial mRNA sequencing from low input samples through ribosomal RNA depletion |
| title_sort | efficient and cost effective bacterial mrna sequencing from low input samples through ribosomal rna depletion |
| topic | Bacterial mRNA sequencing mRNA enrichment rRNA depletion Low input total RNA |
| url | http://link.springer.com/article/10.1186/s12864-020-07134-4 |
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