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Purification of immune-active macrophage super enhancers by chemical cross-linked chromatin immune precipitation

Isolation of extraordinarily long-length super-enhancers (SEs) using typical chromatin immune precipitation (ChIP) techniques can lead to DNA breakage due to uncontrolled cross-linking. We present a redefined ChIP technique for SE purification. After controlled paraformaldehyde-based cross-linking,...

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Detalles Bibliográficos
Autores principales: Das, Sonali, Mukherjee, Sohitri, Ali, Nahid
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Elsevier 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9852664/
https://www.ncbi.nlm.nih.gov/pubmed/36638018
http://dx.doi.org/10.1016/j.xpro.2022.102004
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author Das, Sonali
Mukherjee, Sohitri
Ali, Nahid
author_facet Das, Sonali
Mukherjee, Sohitri
Ali, Nahid
author_sort Das, Sonali
collection PubMed
description Isolation of extraordinarily long-length super-enhancers (SEs) using typical chromatin immune precipitation (ChIP) techniques can lead to DNA breakage due to uncontrolled cross-linking. We present a redefined ChIP technique for SE purification. After controlled paraformaldehyde-based cross-linking, glycine was used to quench the cross-linker followed by mild sonication. The sonication produced ideal fragment length of long-length SE chromatin. Presently, miR146a-5p SE of macrophages was pulled using BRD4 protein. Our protocol can reproducibly simplify the SE element isolation issues, in a quality-controlled manner. For complete details on the use and execution of this protocol, please refer to Das et al. (2021).(1)
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spelling pubmed-98526642023-01-21 Purification of immune-active macrophage super enhancers by chemical cross-linked chromatin immune precipitation Das, Sonali Mukherjee, Sohitri Ali, Nahid STAR Protoc Protocol Isolation of extraordinarily long-length super-enhancers (SEs) using typical chromatin immune precipitation (ChIP) techniques can lead to DNA breakage due to uncontrolled cross-linking. We present a redefined ChIP technique for SE purification. After controlled paraformaldehyde-based cross-linking, glycine was used to quench the cross-linker followed by mild sonication. The sonication produced ideal fragment length of long-length SE chromatin. Presently, miR146a-5p SE of macrophages was pulled using BRD4 protein. Our protocol can reproducibly simplify the SE element isolation issues, in a quality-controlled manner. For complete details on the use and execution of this protocol, please refer to Das et al. (2021).(1) Elsevier 2023-01-11 /pmc/articles/PMC9852664/ /pubmed/36638018 http://dx.doi.org/10.1016/j.xpro.2022.102004 Text en © 2022 The Authors 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
Das, Sonali
Mukherjee, Sohitri
Ali, Nahid
Purification of immune-active macrophage super enhancers by chemical cross-linked chromatin immune precipitation
title Purification of immune-active macrophage super enhancers by chemical cross-linked chromatin immune precipitation
title_full Purification of immune-active macrophage super enhancers by chemical cross-linked chromatin immune precipitation
title_fullStr Purification of immune-active macrophage super enhancers by chemical cross-linked chromatin immune precipitation
title_full_unstemmed Purification of immune-active macrophage super enhancers by chemical cross-linked chromatin immune precipitation
title_short Purification of immune-active macrophage super enhancers by chemical cross-linked chromatin immune precipitation
title_sort purification of immune-active macrophage super enhancers by chemical cross-linked chromatin immune precipitation
topic Protocol
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9852664/
https://www.ncbi.nlm.nih.gov/pubmed/36638018
http://dx.doi.org/10.1016/j.xpro.2022.102004
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AT alinahid purificationofimmuneactivemacrophagesuperenhancersbychemicalcrosslinkedchromatinimmuneprecipitation