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RevGel-seq: instrument-free single-cell RNA sequencing using a reversible hydrogel for cell-specific barcoding
Progress in sample preparation for scRNA-seq is reported based on RevGel-seq, a reversible-hydrogel technology optimized for samples of fresh cells. Complexes of one cell paired with one barcoded bead are stabilized by a chemical linker and dispersed in a hydrogel in the liquid state. Upon gelation...
Autores principales: | , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Nature Publishing Group UK
2023
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10039079/ https://www.ncbi.nlm.nih.gov/pubmed/36964177 http://dx.doi.org/10.1038/s41598-023-31915-y |
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author | Komatsu, Jun Cico, Alba Poncin, Raya Le Bohec, Maël Morf, Jörg Lipin, Stanislav Graindorge, Antoine Eckert, Hélène Saffarian, Azadeh Cathaly, Léa Guérin, Frédéric Majello, Sara Ulveling, Damien Vayaboury, Anaïs Fernandez, Nicolas Dimitrova, Dilyana Bussell, Xavier Fourne, Yannick Chaumat, Pierre André, Barbara Baldivia, Elodie Godet, Ulysse Guinin, Mathieu Moretto, Vivien Ismail, Joy Caille, Olivier Roblot, Natacha Beaupère, Carine Liboz, Alexandrine Guillemain, Ghislaine Blondeau, Bertrand Walrafen, Pierre Edelstein, Stuart |
author_facet | Komatsu, Jun Cico, Alba Poncin, Raya Le Bohec, Maël Morf, Jörg Lipin, Stanislav Graindorge, Antoine Eckert, Hélène Saffarian, Azadeh Cathaly, Léa Guérin, Frédéric Majello, Sara Ulveling, Damien Vayaboury, Anaïs Fernandez, Nicolas Dimitrova, Dilyana Bussell, Xavier Fourne, Yannick Chaumat, Pierre André, Barbara Baldivia, Elodie Godet, Ulysse Guinin, Mathieu Moretto, Vivien Ismail, Joy Caille, Olivier Roblot, Natacha Beaupère, Carine Liboz, Alexandrine Guillemain, Ghislaine Blondeau, Bertrand Walrafen, Pierre Edelstein, Stuart |
author_sort | Komatsu, Jun |
collection | PubMed |
description | Progress in sample preparation for scRNA-seq is reported based on RevGel-seq, a reversible-hydrogel technology optimized for samples of fresh cells. Complexes of one cell paired with one barcoded bead are stabilized by a chemical linker and dispersed in a hydrogel in the liquid state. Upon gelation on ice the complexes are immobilized and physically separated without requiring nanowells or droplets. Cell lysis is triggered by detergent diffusion, and RNA molecules are captured on the adjacent barcoded beads for further processing with reverse transcription and preparation for cDNA sequencing. As a proof of concept, analysis of PBMC using RevGel-seq achieves results similar to microfluidic-based technologies when using the same original sample and the same data analysis software. In addition, a clinically relevant application of RevGel-seq is presented for pancreatic islet cells. Furthermore, characterizations carried out on cardiomyocytes demonstrate that the hydrogel technology readily accommodates very large cells. Standard analyses are in the 10,000-input cell range with the current gelation device, in order to satisfy common requirements for single-cell research. A convenient stopping point after two hours has been established by freezing at the cell lysis step, with full preservation of gene expression profiles. Overall, our results show that RevGel-seq represents an accessible and efficient instrument-free alternative, enabling flexibility in terms of experimental design and timing of sample processing, while providing broad coverage of cell types. |
format | Online Article Text |
id | pubmed-10039079 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | Nature Publishing Group UK |
record_format | MEDLINE/PubMed |
spelling | pubmed-100390792023-03-26 RevGel-seq: instrument-free single-cell RNA sequencing using a reversible hydrogel for cell-specific barcoding Komatsu, Jun Cico, Alba Poncin, Raya Le Bohec, Maël Morf, Jörg Lipin, Stanislav Graindorge, Antoine Eckert, Hélène Saffarian, Azadeh Cathaly, Léa Guérin, Frédéric Majello, Sara Ulveling, Damien Vayaboury, Anaïs Fernandez, Nicolas Dimitrova, Dilyana Bussell, Xavier Fourne, Yannick Chaumat, Pierre André, Barbara Baldivia, Elodie Godet, Ulysse Guinin, Mathieu Moretto, Vivien Ismail, Joy Caille, Olivier Roblot, Natacha Beaupère, Carine Liboz, Alexandrine Guillemain, Ghislaine Blondeau, Bertrand Walrafen, Pierre Edelstein, Stuart Sci Rep Article Progress in sample preparation for scRNA-seq is reported based on RevGel-seq, a reversible-hydrogel technology optimized for samples of fresh cells. Complexes of one cell paired with one barcoded bead are stabilized by a chemical linker and dispersed in a hydrogel in the liquid state. Upon gelation on ice the complexes are immobilized and physically separated without requiring nanowells or droplets. Cell lysis is triggered by detergent diffusion, and RNA molecules are captured on the adjacent barcoded beads for further processing with reverse transcription and preparation for cDNA sequencing. As a proof of concept, analysis of PBMC using RevGel-seq achieves results similar to microfluidic-based technologies when using the same original sample and the same data analysis software. In addition, a clinically relevant application of RevGel-seq is presented for pancreatic islet cells. Furthermore, characterizations carried out on cardiomyocytes demonstrate that the hydrogel technology readily accommodates very large cells. Standard analyses are in the 10,000-input cell range with the current gelation device, in order to satisfy common requirements for single-cell research. A convenient stopping point after two hours has been established by freezing at the cell lysis step, with full preservation of gene expression profiles. Overall, our results show that RevGel-seq represents an accessible and efficient instrument-free alternative, enabling flexibility in terms of experimental design and timing of sample processing, while providing broad coverage of cell types. Nature Publishing Group UK 2023-03-24 /pmc/articles/PMC10039079/ /pubmed/36964177 http://dx.doi.org/10.1038/s41598-023-31915-y Text en © The Author(s) 2023 https://creativecommons.org/licenses/by/4.0/Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) . |
spellingShingle | Article Komatsu, Jun Cico, Alba Poncin, Raya Le Bohec, Maël Morf, Jörg Lipin, Stanislav Graindorge, Antoine Eckert, Hélène Saffarian, Azadeh Cathaly, Léa Guérin, Frédéric Majello, Sara Ulveling, Damien Vayaboury, Anaïs Fernandez, Nicolas Dimitrova, Dilyana Bussell, Xavier Fourne, Yannick Chaumat, Pierre André, Barbara Baldivia, Elodie Godet, Ulysse Guinin, Mathieu Moretto, Vivien Ismail, Joy Caille, Olivier Roblot, Natacha Beaupère, Carine Liboz, Alexandrine Guillemain, Ghislaine Blondeau, Bertrand Walrafen, Pierre Edelstein, Stuart RevGel-seq: instrument-free single-cell RNA sequencing using a reversible hydrogel for cell-specific barcoding |
title | RevGel-seq: instrument-free single-cell RNA sequencing using a reversible hydrogel for cell-specific barcoding |
title_full | RevGel-seq: instrument-free single-cell RNA sequencing using a reversible hydrogel for cell-specific barcoding |
title_fullStr | RevGel-seq: instrument-free single-cell RNA sequencing using a reversible hydrogel for cell-specific barcoding |
title_full_unstemmed | RevGel-seq: instrument-free single-cell RNA sequencing using a reversible hydrogel for cell-specific barcoding |
title_short | RevGel-seq: instrument-free single-cell RNA sequencing using a reversible hydrogel for cell-specific barcoding |
title_sort | revgel-seq: instrument-free single-cell rna sequencing using a reversible hydrogel for cell-specific barcoding |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10039079/ https://www.ncbi.nlm.nih.gov/pubmed/36964177 http://dx.doi.org/10.1038/s41598-023-31915-y |
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