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An optimized approach for multiplexing single-nuclear ATAC-seq using oligonucleotide-conjugated antibodies
BACKGROUND: Single-cell technologies to analyze transcription and chromatin structure have been widely used in many research areas to reveal the functions and molecular properties of cells at single-cell resolution. Sample multiplexing techniques are valuable when performing single-cell analysis, re...
Autores principales: | , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2023
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10142415/ https://www.ncbi.nlm.nih.gov/pubmed/37118773 http://dx.doi.org/10.1186/s13072-023-00486-7 |
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author | Bera, Betelehem Solomon Thompson, Taylor V. Sosa, Eric Nomaru, Hiroko Reynolds, David Dubin, Robert A. Maqbool, Shahina B. Zheng, Deyou Morrow, Bernice E. Greally, John M. Suzuki, Masako |
author_facet | Bera, Betelehem Solomon Thompson, Taylor V. Sosa, Eric Nomaru, Hiroko Reynolds, David Dubin, Robert A. Maqbool, Shahina B. Zheng, Deyou Morrow, Bernice E. Greally, John M. Suzuki, Masako |
author_sort | Bera, Betelehem Solomon |
collection | PubMed |
description | BACKGROUND: Single-cell technologies to analyze transcription and chromatin structure have been widely used in many research areas to reveal the functions and molecular properties of cells at single-cell resolution. Sample multiplexing techniques are valuable when performing single-cell analysis, reducing technical variation and permitting cost efficiencies. Several commercially available methods have been used in many scRNA-seq studies. On the other hand, while several methods have been published, multiplexing techniques for single nuclear assay for transposase-accessible chromatin (snATAC)-seq assays remain under development. We developed a simple nucleus hashing method using oligonucleotide-conjugated antibodies recognizing nuclear pore complex proteins, NuHash, to perform snATAC-seq library preparations by multiplexing. RESULTS: We performed multiplexing snATAC-seq analyses on a mixture of human and mouse cell samples (two samples, 2-plex, and four samples, 4-plex) using NuHash. The analyses on nuclei with at least 10,000 read counts showed that the demultiplexing accuracy of NuHash was high, and only ten out of 9144 nuclei (2-plex) and 150 of 12,208 nuclei (4-plex) had discordant classifications between NuHash demultiplexing and discrimination using reference genome alignments. The differential open chromatin region (OCR) analysis between female and male samples revealed that male-specific OCRs were enriched in chromosome Y (four out of nine). We also found that five female-specific OCRs (20 OCRs) were on chromosome X. A comparative analysis between snATAC-seq and deeply sequenced bulk ATAC-seq on the same samples revealed that the bulk ATAC-seq signal intensity was positively correlated with the number of cell clusters detected in snATAC-seq. Moreover, when we categorized snATAC-seq peaks based on the number of cell clusters in which the peak was present, we observed different distributions over different genomic features between the groups. This result suggests that the peak intensities of bulk ATAC-seq can be used to identify different types of functional loci. CONCLUSIONS: Our multiplexing method using oligo-conjugated anti-nuclear pore complex proteins, NuHash, permits high-accuracy demultiplexing of samples. The NuHash protocol is straightforward, works on frozen samples, and requires no modifications for snATAC-seq library preparation. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s13072-023-00486-7. |
format | Online Article Text |
id | pubmed-10142415 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-101424152023-04-29 An optimized approach for multiplexing single-nuclear ATAC-seq using oligonucleotide-conjugated antibodies Bera, Betelehem Solomon Thompson, Taylor V. Sosa, Eric Nomaru, Hiroko Reynolds, David Dubin, Robert A. Maqbool, Shahina B. Zheng, Deyou Morrow, Bernice E. Greally, John M. Suzuki, Masako Epigenetics Chromatin Methodology BACKGROUND: Single-cell technologies to analyze transcription and chromatin structure have been widely used in many research areas to reveal the functions and molecular properties of cells at single-cell resolution. Sample multiplexing techniques are valuable when performing single-cell analysis, reducing technical variation and permitting cost efficiencies. Several commercially available methods have been used in many scRNA-seq studies. On the other hand, while several methods have been published, multiplexing techniques for single nuclear assay for transposase-accessible chromatin (snATAC)-seq assays remain under development. We developed a simple nucleus hashing method using oligonucleotide-conjugated antibodies recognizing nuclear pore complex proteins, NuHash, to perform snATAC-seq library preparations by multiplexing. RESULTS: We performed multiplexing snATAC-seq analyses on a mixture of human and mouse cell samples (two samples, 2-plex, and four samples, 4-plex) using NuHash. The analyses on nuclei with at least 10,000 read counts showed that the demultiplexing accuracy of NuHash was high, and only ten out of 9144 nuclei (2-plex) and 150 of 12,208 nuclei (4-plex) had discordant classifications between NuHash demultiplexing and discrimination using reference genome alignments. The differential open chromatin region (OCR) analysis between female and male samples revealed that male-specific OCRs were enriched in chromosome Y (four out of nine). We also found that five female-specific OCRs (20 OCRs) were on chromosome X. A comparative analysis between snATAC-seq and deeply sequenced bulk ATAC-seq on the same samples revealed that the bulk ATAC-seq signal intensity was positively correlated with the number of cell clusters detected in snATAC-seq. Moreover, when we categorized snATAC-seq peaks based on the number of cell clusters in which the peak was present, we observed different distributions over different genomic features between the groups. This result suggests that the peak intensities of bulk ATAC-seq can be used to identify different types of functional loci. CONCLUSIONS: Our multiplexing method using oligo-conjugated anti-nuclear pore complex proteins, NuHash, permits high-accuracy demultiplexing of samples. The NuHash protocol is straightforward, works on frozen samples, and requires no modifications for snATAC-seq library preparation. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s13072-023-00486-7. BioMed Central 2023-04-28 /pmc/articles/PMC10142415/ /pubmed/37118773 http://dx.doi.org/10.1186/s13072-023-00486-7 Text en © The Author(s) 2023 https://creativecommons.org/licenses/by/4.0/Open AccessThis 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/) . The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/ (https://creativecommons.org/publicdomain/zero/1.0/) ) applies to the data made available in this article, unless otherwise stated in a credit line to the data. |
spellingShingle | Methodology Bera, Betelehem Solomon Thompson, Taylor V. Sosa, Eric Nomaru, Hiroko Reynolds, David Dubin, Robert A. Maqbool, Shahina B. Zheng, Deyou Morrow, Bernice E. Greally, John M. Suzuki, Masako An optimized approach for multiplexing single-nuclear ATAC-seq using oligonucleotide-conjugated antibodies |
title | An optimized approach for multiplexing single-nuclear ATAC-seq using oligonucleotide-conjugated antibodies |
title_full | An optimized approach for multiplexing single-nuclear ATAC-seq using oligonucleotide-conjugated antibodies |
title_fullStr | An optimized approach for multiplexing single-nuclear ATAC-seq using oligonucleotide-conjugated antibodies |
title_full_unstemmed | An optimized approach for multiplexing single-nuclear ATAC-seq using oligonucleotide-conjugated antibodies |
title_short | An optimized approach for multiplexing single-nuclear ATAC-seq using oligonucleotide-conjugated antibodies |
title_sort | optimized approach for multiplexing single-nuclear atac-seq using oligonucleotide-conjugated antibodies |
topic | Methodology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10142415/ https://www.ncbi.nlm.nih.gov/pubmed/37118773 http://dx.doi.org/10.1186/s13072-023-00486-7 |
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