Effect of methanol fixation on single-cell RNA sequencing of the murine dentate gyrus
Single-cell RNA sequencing (scRNA-seq) provides a powerful tool to evaluate the transcriptomic landscape and heterogeneity of thousands of cells in parallel. However, complex study designs or the unavailability of in-house instruments require the temporal disconnection between sample preparation and...
| Main Authors: | , , , , , , , |
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2023-10-01
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| Series: | Frontiers in Molecular Neuroscience |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fnmol.2023.1223798/full |
| _version_ | 1797666518317662208 |
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| author | Marta Sánchez-Carbonell Patricia Jiménez Peinado Cathrin Bayer-Kaufmann Jean-Christopher Hennings Yvonne Hofmann Silvio Schmidt Silvio Schmidt Silvio Schmidt Otto W. Witte Otto W. Witte Otto W. Witte Anja Urbach Anja Urbach Anja Urbach |
| author_facet | Marta Sánchez-Carbonell Patricia Jiménez Peinado Cathrin Bayer-Kaufmann Jean-Christopher Hennings Yvonne Hofmann Silvio Schmidt Silvio Schmidt Silvio Schmidt Otto W. Witte Otto W. Witte Otto W. Witte Anja Urbach Anja Urbach Anja Urbach |
| author_sort | Marta Sánchez-Carbonell |
| collection | DOAJ |
| description | Single-cell RNA sequencing (scRNA-seq) provides a powerful tool to evaluate the transcriptomic landscape and heterogeneity of thousands of cells in parallel. However, complex study designs or the unavailability of in-house instruments require the temporal disconnection between sample preparation and library construction, raising the need for efficient sample preservation methods which are compatible with scRNA-seq downstream analysis. Several studies evaluated the effect of methanol fixation as preservation method, yet none of them deeply assessed its effect on adult primary dissociated brain tissue. Here, we evaluated its effect on murine dentate gyrus (DG) single cell suspensions and on subsequent scRNA-seq downstream analysis by performing SOrting and Robot-assisted Transcriptome SEQuencing (SORT-seq), a partially robotized version of the CEL-seq2 protocol. Our results show that MeOH fixation preserves RNA integrity and has no apparent effects on cDNA library construction. They also suggest that fixation protects from sorting-induced cell stress and increases the proportion of high-quality cells. Despite evidence of mRNA leakage in fixed cells, their relative gene expression levels correlate well with those of fresh cells and fixation does not significantly affect the variance of the dataset. Moreover, it allows the identification of all major DG cell populations, including neural precursors, granule neurons and different glial cell types, with a tendency to preserve more neurons that are underrepresented in fresh samples. Overall, our data show that MeOH fixation is suitable for preserving primary neural cells for subsequent single-cell RNA profiling, helping to overcome challenges arising from complex workflows, improve experimental flexibility and facilitate scientific collaboration. |
| first_indexed | 2024-03-11T20:00:31Z |
| format | Article |
| id | doaj.art-7a5bc465502c4afbb0f6f3897d5f453e |
| institution | Directory Open Access Journal |
| issn | 1662-5099 |
| language | English |
| last_indexed | 2024-03-11T20:00:31Z |
| publishDate | 2023-10-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Molecular Neuroscience |
| spelling | doaj.art-7a5bc465502c4afbb0f6f3897d5f453e2023-10-04T08:37:30ZengFrontiers Media S.A.Frontiers in Molecular Neuroscience1662-50992023-10-011610.3389/fnmol.2023.12237981223798Effect of methanol fixation on single-cell RNA sequencing of the murine dentate gyrusMarta Sánchez-Carbonell0Patricia Jiménez Peinado1Cathrin Bayer-Kaufmann2Jean-Christopher Hennings3Yvonne Hofmann4Silvio Schmidt5Silvio Schmidt6Silvio Schmidt7Otto W. Witte8Otto W. Witte9Otto W. Witte10Anja Urbach11Anja Urbach12Anja Urbach13Department of Neurology, Jena University Hospital, Jena, GermanyDepartment of Neurology, Jena University Hospital, Jena, GermanyDepartment of Neurology, Jena University Hospital, Jena, GermanyInstitute of Human Genetics, Jena University Hospital, Jena, GermanyDepartment of Internal Medicine V, Jena University Hospital, Jena, GermanyDepartment of Neurology, Jena University Hospital, Jena, GermanyJena Center for Healthy Aging, Jena University Hospital, Jena, GermanyBrain Imaging Center, Jena University Hospital, Jena, GermanyDepartment of Neurology, Jena University Hospital, Jena, GermanyJena Center for Healthy Aging, Jena University Hospital, Jena, GermanyAging Research Center (ARC) Jena, Jena, GermanyDepartment of Neurology, Jena University Hospital, Jena, GermanyJena Center for Healthy Aging, Jena University Hospital, Jena, GermanyAging Research Center (ARC) Jena, Jena, GermanySingle-cell RNA sequencing (scRNA-seq) provides a powerful tool to evaluate the transcriptomic landscape and heterogeneity of thousands of cells in parallel. However, complex study designs or the unavailability of in-house instruments require the temporal disconnection between sample preparation and library construction, raising the need for efficient sample preservation methods which are compatible with scRNA-seq downstream analysis. Several studies evaluated the effect of methanol fixation as preservation method, yet none of them deeply assessed its effect on adult primary dissociated brain tissue. Here, we evaluated its effect on murine dentate gyrus (DG) single cell suspensions and on subsequent scRNA-seq downstream analysis by performing SOrting and Robot-assisted Transcriptome SEQuencing (SORT-seq), a partially robotized version of the CEL-seq2 protocol. Our results show that MeOH fixation preserves RNA integrity and has no apparent effects on cDNA library construction. They also suggest that fixation protects from sorting-induced cell stress and increases the proportion of high-quality cells. Despite evidence of mRNA leakage in fixed cells, their relative gene expression levels correlate well with those of fresh cells and fixation does not significantly affect the variance of the dataset. Moreover, it allows the identification of all major DG cell populations, including neural precursors, granule neurons and different glial cell types, with a tendency to preserve more neurons that are underrepresented in fresh samples. Overall, our data show that MeOH fixation is suitable for preserving primary neural cells for subsequent single-cell RNA profiling, helping to overcome challenges arising from complex workflows, improve experimental flexibility and facilitate scientific collaboration.https://www.frontiersin.org/articles/10.3389/fnmol.2023.1223798/fullmethanol fixationprimary neural cellsSORT-seqdentate gyrusadult neurogenesissingle-cell RNA sequencing |
| spellingShingle | Marta Sánchez-Carbonell Patricia Jiménez Peinado Cathrin Bayer-Kaufmann Jean-Christopher Hennings Yvonne Hofmann Silvio Schmidt Silvio Schmidt Silvio Schmidt Otto W. Witte Otto W. Witte Otto W. Witte Anja Urbach Anja Urbach Anja Urbach Effect of methanol fixation on single-cell RNA sequencing of the murine dentate gyrus Frontiers in Molecular Neuroscience methanol fixation primary neural cells SORT-seq dentate gyrus adult neurogenesis single-cell RNA sequencing |
| title | Effect of methanol fixation on single-cell RNA sequencing of the murine dentate gyrus |
| title_full | Effect of methanol fixation on single-cell RNA sequencing of the murine dentate gyrus |
| title_fullStr | Effect of methanol fixation on single-cell RNA sequencing of the murine dentate gyrus |
| title_full_unstemmed | Effect of methanol fixation on single-cell RNA sequencing of the murine dentate gyrus |
| title_short | Effect of methanol fixation on single-cell RNA sequencing of the murine dentate gyrus |
| title_sort | effect of methanol fixation on single cell rna sequencing of the murine dentate gyrus |
| topic | methanol fixation primary neural cells SORT-seq dentate gyrus adult neurogenesis single-cell RNA sequencing |
| url | https://www.frontiersin.org/articles/10.3389/fnmol.2023.1223798/full |
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