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Preparation of optimized concanavalin A-conjugated Dynabeads® magnetic beads for CUT&Tag
Epigenome research has employed various methods to identify the genomic location of proteins of interest, such as transcription factors and histone modifications. A recently established method called CUT&Tag uses a Protein-A Tn5 transposase fusion protein, which cuts the genome and inserts adapt...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2021
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8594809/ https://www.ncbi.nlm.nih.gov/pubmed/34784358 http://dx.doi.org/10.1371/journal.pone.0259846 |
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author | Fujiwara, Yasuhiro Tanno, Yuji Sugishita, Hiroki Kishi, Yusuke Makino, Yoshinori Okada, Yuki |
author_facet | Fujiwara, Yasuhiro Tanno, Yuji Sugishita, Hiroki Kishi, Yusuke Makino, Yoshinori Okada, Yuki |
author_sort | Fujiwara, Yasuhiro |
collection | PubMed |
description | Epigenome research has employed various methods to identify the genomic location of proteins of interest, such as transcription factors and histone modifications. A recently established method called CUT&Tag uses a Protein-A Tn5 transposase fusion protein, which cuts the genome and inserts adapter sequences nearby the target protein. Throughout most of the CUT&Tag procedure, cells are held on concanavalin A (con A)-conjugated magnetic beads. Proper holding of cells would be decisive for the accessibility of Tn5 to the chromatin, and efficacy of the procedure of washing cells. However, BioMag®Plus ConA magnetic beads, used in the original CUT&Tag protocol, often exhibit poor suspendability and severe aggregation. Here, we compared the BioMag beads and Dynabeads® magnetic particles of which conjugation of con A was done by our hands, and examined the performance of these magnetic beads in CUT&Tag. Among tested, one of the Dynabeads, MyOne-T1, kept excessive suspendability in a buffer even after overnight incubation. Furthermore, the MyOne-T1 beads notably improved the sensitivity in CUT&Tag assay for H3K4me3. In conclusion, the arrangement and the selection of MyOne-T1 refine the suspendability of beads, which improves the association of chromatin with Tn5, which enhances the sensitivity in CUT&Tag assay. |
format | Online Article Text |
id | pubmed-8594809 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-85948092021-11-17 Preparation of optimized concanavalin A-conjugated Dynabeads® magnetic beads for CUT&Tag Fujiwara, Yasuhiro Tanno, Yuji Sugishita, Hiroki Kishi, Yusuke Makino, Yoshinori Okada, Yuki PLoS One Research Article Epigenome research has employed various methods to identify the genomic location of proteins of interest, such as transcription factors and histone modifications. A recently established method called CUT&Tag uses a Protein-A Tn5 transposase fusion protein, which cuts the genome and inserts adapter sequences nearby the target protein. Throughout most of the CUT&Tag procedure, cells are held on concanavalin A (con A)-conjugated magnetic beads. Proper holding of cells would be decisive for the accessibility of Tn5 to the chromatin, and efficacy of the procedure of washing cells. However, BioMag®Plus ConA magnetic beads, used in the original CUT&Tag protocol, often exhibit poor suspendability and severe aggregation. Here, we compared the BioMag beads and Dynabeads® magnetic particles of which conjugation of con A was done by our hands, and examined the performance of these magnetic beads in CUT&Tag. Among tested, one of the Dynabeads, MyOne-T1, kept excessive suspendability in a buffer even after overnight incubation. Furthermore, the MyOne-T1 beads notably improved the sensitivity in CUT&Tag assay for H3K4me3. In conclusion, the arrangement and the selection of MyOne-T1 refine the suspendability of beads, which improves the association of chromatin with Tn5, which enhances the sensitivity in CUT&Tag assay. Public Library of Science 2021-11-16 /pmc/articles/PMC8594809/ /pubmed/34784358 http://dx.doi.org/10.1371/journal.pone.0259846 Text en © 2021 Fujiwara et al https://creativecommons.org/licenses/by/4.0/This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. |
spellingShingle | Research Article Fujiwara, Yasuhiro Tanno, Yuji Sugishita, Hiroki Kishi, Yusuke Makino, Yoshinori Okada, Yuki Preparation of optimized concanavalin A-conjugated Dynabeads® magnetic beads for CUT&Tag |
title | Preparation of optimized concanavalin A-conjugated Dynabeads® magnetic beads for CUT&Tag |
title_full | Preparation of optimized concanavalin A-conjugated Dynabeads® magnetic beads for CUT&Tag |
title_fullStr | Preparation of optimized concanavalin A-conjugated Dynabeads® magnetic beads for CUT&Tag |
title_full_unstemmed | Preparation of optimized concanavalin A-conjugated Dynabeads® magnetic beads for CUT&Tag |
title_short | Preparation of optimized concanavalin A-conjugated Dynabeads® magnetic beads for CUT&Tag |
title_sort | preparation of optimized concanavalin a-conjugated dynabeads® magnetic beads for cut&tag |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8594809/ https://www.ncbi.nlm.nih.gov/pubmed/34784358 http://dx.doi.org/10.1371/journal.pone.0259846 |
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