Using single-worm RNA sequencing to study C. elegans responses to pathogen infection
Abstract Background Caenorhabditis elegans is an excellent research model whose populations have been used in many studies to address various biological questions. Although worm-to-worm phenotypic variations in isogenic populations have been persistently observed, they are not well understood and ar...
| Main Authors: | , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
BMC
2022-09-01
|
| Series: | BMC Genomics |
| Subjects: | |
| Online Access: | https://doi.org/10.1186/s12864-022-08878-x |
| _version_ | 1818020409615843328 |
|---|---|
| author | Archer J. Wang Phillip Wibisono Blake M. Geppert Yiyong Liu |
| author_facet | Archer J. Wang Phillip Wibisono Blake M. Geppert Yiyong Liu |
| author_sort | Archer J. Wang |
| collection | DOAJ |
| description | Abstract Background Caenorhabditis elegans is an excellent research model whose populations have been used in many studies to address various biological questions. Although worm-to-worm phenotypic variations in isogenic populations have been persistently observed, they are not well understood and are often ignored or averaged out in studies, masking the impacts of such variations on data collection and interpretation. Single-worm RNA sequencing that profiles the transcriptomes of individual animals has the power to examine differences between individuals in a worm population, but this approach has been understudied. The integrity of the starting RNA, the quality of the library and sequence data, as well as the transcriptome-profiling effectiveness of single-worm RNA-seq remain unclear. Therefore, more studies are needed to improve this technique and its application in research. Results In this study, we aimed to develop a single-worm RNA-seq method that includes five steps: worm lysis and RNA extraction, cDNA synthesis, library preparation, sequencing, and sequence data analysis. We found that the mechanical lysis of worms using a Qiagen TissueLyser maintained RNA integrity and determined that the quality of our single-worm libraries was comparable to that of standard RNA-seq libraries based on assessments of a variety of parameters. Furthermore, analysis of pathogen infection-induced gene expression using single-worm RNA-seq identified a core set of genes and biological processes relating to the immune response and metabolism affected by infection. These results demonstrate the effectiveness of our single-worm RNA-seq method in transcriptome profiling and its usefulness in addressing biological questions. Conclusions We have developed a single-worm RNA-seq method to effectively profile gene expression in individual C. elegans and have applied this method to study C. elegans responses to pathogen infection. Key aspects of our single-worm RNA-seq libraries were comparable to those of standard RNA-seq libraries. The single-worm method captured the core set of, but not all, infection-affected genes and biological processes revealed by the standard method, indicating that there was gene regulation that is not shared by all individuals in a population. Our study suggests that combining single-worm and standard RNA-seq approaches will allow for detecting and distinguishing shared and individual-specific gene activities in isogenic populations. |
| first_indexed | 2024-04-14T08:04:32Z |
| format | Article |
| id | doaj.art-cb16a3aba66d44048811d6e5c9271b50 |
| institution | Directory Open Access Journal |
| issn | 1471-2164 |
| language | English |
| last_indexed | 2024-04-14T08:04:32Z |
| publishDate | 2022-09-01 |
| publisher | BMC |
| record_format | Article |
| series | BMC Genomics |
| spelling | doaj.art-cb16a3aba66d44048811d6e5c9271b502022-12-22T02:04:47ZengBMCBMC Genomics1471-21642022-09-0123111610.1186/s12864-022-08878-xUsing single-worm RNA sequencing to study C. elegans responses to pathogen infectionArcher J. Wang0Phillip Wibisono1Blake M. Geppert2Yiyong Liu3Genomics Core, Washington State UniversityDepartment of Translational Medicine & Physiology, Elson S. Floyd College of Medicine, Washington State UniversityGenomics Core, Washington State UniversityGenomics Core, Washington State UniversityAbstract Background Caenorhabditis elegans is an excellent research model whose populations have been used in many studies to address various biological questions. Although worm-to-worm phenotypic variations in isogenic populations have been persistently observed, they are not well understood and are often ignored or averaged out in studies, masking the impacts of such variations on data collection and interpretation. Single-worm RNA sequencing that profiles the transcriptomes of individual animals has the power to examine differences between individuals in a worm population, but this approach has been understudied. The integrity of the starting RNA, the quality of the library and sequence data, as well as the transcriptome-profiling effectiveness of single-worm RNA-seq remain unclear. Therefore, more studies are needed to improve this technique and its application in research. Results In this study, we aimed to develop a single-worm RNA-seq method that includes five steps: worm lysis and RNA extraction, cDNA synthesis, library preparation, sequencing, and sequence data analysis. We found that the mechanical lysis of worms using a Qiagen TissueLyser maintained RNA integrity and determined that the quality of our single-worm libraries was comparable to that of standard RNA-seq libraries based on assessments of a variety of parameters. Furthermore, analysis of pathogen infection-induced gene expression using single-worm RNA-seq identified a core set of genes and biological processes relating to the immune response and metabolism affected by infection. These results demonstrate the effectiveness of our single-worm RNA-seq method in transcriptome profiling and its usefulness in addressing biological questions. Conclusions We have developed a single-worm RNA-seq method to effectively profile gene expression in individual C. elegans and have applied this method to study C. elegans responses to pathogen infection. Key aspects of our single-worm RNA-seq libraries were comparable to those of standard RNA-seq libraries. The single-worm method captured the core set of, but not all, infection-affected genes and biological processes revealed by the standard method, indicating that there was gene regulation that is not shared by all individuals in a population. Our study suggests that combining single-worm and standard RNA-seq approaches will allow for detecting and distinguishing shared and individual-specific gene activities in isogenic populations.https://doi.org/10.1186/s12864-022-08878-xSingle-worm RNA sequencingLow-input RNA sequencingLibrary preparationLibrary quality metricsPathogen infectionCaenorhabditis elegans |
| spellingShingle | Archer J. Wang Phillip Wibisono Blake M. Geppert Yiyong Liu Using single-worm RNA sequencing to study C. elegans responses to pathogen infection BMC Genomics Single-worm RNA sequencing Low-input RNA sequencing Library preparation Library quality metrics Pathogen infection Caenorhabditis elegans |
| title | Using single-worm RNA sequencing to study C. elegans responses to pathogen infection |
| title_full | Using single-worm RNA sequencing to study C. elegans responses to pathogen infection |
| title_fullStr | Using single-worm RNA sequencing to study C. elegans responses to pathogen infection |
| title_full_unstemmed | Using single-worm RNA sequencing to study C. elegans responses to pathogen infection |
| title_short | Using single-worm RNA sequencing to study C. elegans responses to pathogen infection |
| title_sort | using single worm rna sequencing to study c elegans responses to pathogen infection |
| topic | Single-worm RNA sequencing Low-input RNA sequencing Library preparation Library quality metrics Pathogen infection Caenorhabditis elegans |
| url | https://doi.org/10.1186/s12864-022-08878-x |
| work_keys_str_mv | AT archerjwang usingsinglewormrnasequencingtostudycelegansresponsestopathogeninfection AT phillipwibisono usingsinglewormrnasequencingtostudycelegansresponsestopathogeninfection AT blakemgeppert usingsinglewormrnasequencingtostudycelegansresponsestopathogeninfection AT yiyongliu usingsinglewormrnasequencingtostudycelegansresponsestopathogeninfection |