Protocol to evaluate cell lineage stability of mouse natural and induced regulatory T cells using bisulfite sequencing

The establishment of regulatory T cells (Treg)-specific demethylation regions (TSDRs) is essential for the Treg-lineage stability. Here, we present a protocol using bisulfite sequencing to assess Treg-lineage stability. The protocol describes the isolation of lymphocytes and DNA extraction, followed...

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Autores principales: Arai, Masaya, Fukuda, Aine, Morimoto, Reo, Nakamura, Yamami, Ci, Zhaohong, Sakaguchi, Shimon
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Elsevier 2022
Materias:
Protocol
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9489535/
https://www.ncbi.nlm.nih.gov/pubmed/36121747
http://dx.doi.org/10.1016/j.xpro.2022.101694
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author Arai, Masaya
Fukuda, Aine
Morimoto, Reo
Nakamura, Yamami
Ci, Zhaohong
Sakaguchi, Shimon
author_facet Arai, Masaya
Fukuda, Aine
Morimoto, Reo
Nakamura, Yamami
Ci, Zhaohong
Sakaguchi, Shimon
author_sort Arai, Masaya
collection PubMed
description The establishment of regulatory T cells (Treg)-specific demethylation regions (TSDRs) is essential for the Treg-lineage stability. Here, we present a protocol using bisulfite sequencing to assess Treg-lineage stability. The protocol describes the isolation of lymphocytes and DNA extraction, followed by bisulfite conversion in unmethylated CpG DNA, bisulfite PCR and cloning, and sequencing to define the TSDR methylation. This protocol uses lymph nodes and spleen tissues and can be adapted to assess the methylation status of Tregs in other tissue types.
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spelling pubmed-94895352022-09-22 Protocol to evaluate cell lineage stability of mouse natural and induced regulatory T cells using bisulfite sequencing Arai, Masaya Fukuda, Aine Morimoto, Reo Nakamura, Yamami Ci, Zhaohong Sakaguchi, Shimon STAR Protoc Protocol The establishment of regulatory T cells (Treg)-specific demethylation regions (TSDRs) is essential for the Treg-lineage stability. Here, we present a protocol using bisulfite sequencing to assess Treg-lineage stability. The protocol describes the isolation of lymphocytes and DNA extraction, followed by bisulfite conversion in unmethylated CpG DNA, bisulfite PCR and cloning, and sequencing to define the TSDR methylation. This protocol uses lymph nodes and spleen tissues and can be adapted to assess the methylation status of Tregs in other tissue types. Elsevier 2022-09-18 /pmc/articles/PMC9489535/ /pubmed/36121747 http://dx.doi.org/10.1016/j.xpro.2022.101694 Text en © 2022 The Author(s) https://creativecommons.org/licenses/by-nc-nd/4.0/This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
spellingShingle Protocol
Arai, Masaya
Fukuda, Aine
Morimoto, Reo
Nakamura, Yamami
Ci, Zhaohong
Sakaguchi, Shimon
Protocol to evaluate cell lineage stability of mouse natural and induced regulatory T cells using bisulfite sequencing
title Protocol to evaluate cell lineage stability of mouse natural and induced regulatory T cells using bisulfite sequencing
title_full Protocol to evaluate cell lineage stability of mouse natural and induced regulatory T cells using bisulfite sequencing
title_fullStr Protocol to evaluate cell lineage stability of mouse natural and induced regulatory T cells using bisulfite sequencing
title_full_unstemmed Protocol to evaluate cell lineage stability of mouse natural and induced regulatory T cells using bisulfite sequencing
title_short Protocol to evaluate cell lineage stability of mouse natural and induced regulatory T cells using bisulfite sequencing
title_sort protocol to evaluate cell lineage stability of mouse natural and induced regulatory t cells using bisulfite sequencing
topic Protocol
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9489535/
https://www.ncbi.nlm.nih.gov/pubmed/36121747
http://dx.doi.org/10.1016/j.xpro.2022.101694
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