Cargando…
High-throughput single-cell epigenomic profiling by targeted insertion of promoters (TIP-seq)
Chromatin profiling in single cells has been extremely challenging and almost exclusively limited to histone proteins. In cases where single-cell methods have shown promise, many require highly specialized equipment or cell type–specific protocols and are relatively low throughput. Here, we combine...
Autores principales: | , , , , , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Rockefeller University Press
2021
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8600797/ https://www.ncbi.nlm.nih.gov/pubmed/34783858 http://dx.doi.org/10.1083/jcb.202103078 |
_version_ | 1784601224960540672 |
---|---|
author | Bartlett, Daniel A. Dileep, Vishnu Handa, Tetsuya Ohkawa, Yasuyuki Kimura, Hiroshi Henikoff, Steven Gilbert, David M. |
author_facet | Bartlett, Daniel A. Dileep, Vishnu Handa, Tetsuya Ohkawa, Yasuyuki Kimura, Hiroshi Henikoff, Steven Gilbert, David M. |
author_sort | Bartlett, Daniel A. |
collection | PubMed |
description | Chromatin profiling in single cells has been extremely challenging and almost exclusively limited to histone proteins. In cases where single-cell methods have shown promise, many require highly specialized equipment or cell type–specific protocols and are relatively low throughput. Here, we combine the advantages of tagmentation, linear amplification, and combinatorial indexing to produce a high-throughput single-cell DNA binding site mapping method that is simple, inexpensive, and capable of multiplexing several independent samples per experiment. Targeted insertion of promoters sequencing (TIP-seq) uses Tn5 fused to proteinA to insert a T7 RNA polymerase promoter adjacent to a chromatin protein of interest. Linear amplification of flanking DNA with T7 polymerase before sequencing library preparation provides ∼10-fold higher unique reads per single cell compared with other methods. We applied TIP-seq to map histone modifications, RNA polymerase II (RNAPII), and transcription factor CTCF binding sites in single human and mouse cells. |
format | Online Article Text |
id | pubmed-8600797 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | Rockefeller University Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-86007972022-06-06 High-throughput single-cell epigenomic profiling by targeted insertion of promoters (TIP-seq) Bartlett, Daniel A. Dileep, Vishnu Handa, Tetsuya Ohkawa, Yasuyuki Kimura, Hiroshi Henikoff, Steven Gilbert, David M. J Cell Biol Tools Chromatin profiling in single cells has been extremely challenging and almost exclusively limited to histone proteins. In cases where single-cell methods have shown promise, many require highly specialized equipment or cell type–specific protocols and are relatively low throughput. Here, we combine the advantages of tagmentation, linear amplification, and combinatorial indexing to produce a high-throughput single-cell DNA binding site mapping method that is simple, inexpensive, and capable of multiplexing several independent samples per experiment. Targeted insertion of promoters sequencing (TIP-seq) uses Tn5 fused to proteinA to insert a T7 RNA polymerase promoter adjacent to a chromatin protein of interest. Linear amplification of flanking DNA with T7 polymerase before sequencing library preparation provides ∼10-fold higher unique reads per single cell compared with other methods. We applied TIP-seq to map histone modifications, RNA polymerase II (RNAPII), and transcription factor CTCF binding sites in single human and mouse cells. Rockefeller University Press 2021-11-16 /pmc/articles/PMC8600797/ /pubmed/34783858 http://dx.doi.org/10.1083/jcb.202103078 Text en © 2021 Bartlett et al. https://creativecommons.org/licenses/by-nc-sa/4.0/http://www.rupress.org/terms/This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms/). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 International license, as described at https://creativecommons.org/licenses/by-nc-sa/4.0/). |
spellingShingle | Tools Bartlett, Daniel A. Dileep, Vishnu Handa, Tetsuya Ohkawa, Yasuyuki Kimura, Hiroshi Henikoff, Steven Gilbert, David M. High-throughput single-cell epigenomic profiling by targeted insertion of promoters (TIP-seq) |
title | High-throughput single-cell epigenomic profiling by targeted insertion of promoters (TIP-seq) |
title_full | High-throughput single-cell epigenomic profiling by targeted insertion of promoters (TIP-seq) |
title_fullStr | High-throughput single-cell epigenomic profiling by targeted insertion of promoters (TIP-seq) |
title_full_unstemmed | High-throughput single-cell epigenomic profiling by targeted insertion of promoters (TIP-seq) |
title_short | High-throughput single-cell epigenomic profiling by targeted insertion of promoters (TIP-seq) |
title_sort | high-throughput single-cell epigenomic profiling by targeted insertion of promoters (tip-seq) |
topic | Tools |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8600797/ https://www.ncbi.nlm.nih.gov/pubmed/34783858 http://dx.doi.org/10.1083/jcb.202103078 |
work_keys_str_mv | AT bartlettdaniela highthroughputsinglecellepigenomicprofilingbytargetedinsertionofpromoterstipseq AT dileepvishnu highthroughputsinglecellepigenomicprofilingbytargetedinsertionofpromoterstipseq AT handatetsuya highthroughputsinglecellepigenomicprofilingbytargetedinsertionofpromoterstipseq AT ohkawayasuyuki highthroughputsinglecellepigenomicprofilingbytargetedinsertionofpromoterstipseq AT kimurahiroshi highthroughputsinglecellepigenomicprofilingbytargetedinsertionofpromoterstipseq AT henikoffsteven highthroughputsinglecellepigenomicprofilingbytargetedinsertionofpromoterstipseq AT gilbertdavidm highthroughputsinglecellepigenomicprofilingbytargetedinsertionofpromoterstipseq |