Long-read ChIA-PET for base-pair-resolution mapping of haplotype-specific chromatin interactions
Li et al . provide a protocol for long-read ChIA-PET, a technique for mapping chromatin interactions. The longer paired-end tags, which are generated by tagmentation, provide sufficient coverage to determine haplotype-specific chromatin interactions at single-nucleotide resolution. Chromatin interac...
Gespeichert in:
| Veröffentlicht in: | Nature protocols 2017-05, Vol.12 (5), p.899-915 |
|---|---|
| Hauptverfasser: | , , , , , , , , , , |
| Format: | Artikel |
| Sprache: | eng |
| Schlagworte: | |
| Online-Zugang: | Volltext |
| Tags: |
Tag hinzufügen
Keine Tags, Fügen Sie den ersten Tag hinzu!
|
| container_end_page | 915 |
|---|---|
| container_issue | 5 |
| container_start_page | 899 |
| container_title | Nature protocols |
| container_volume | 12 |
| creator | Li, Xingwang Luo, Oscar Junhong Wang, Ping Zheng, Meizhen Wang, Danjuan Piecuch, Emaly Zhu, Jacqueline Jufen Tian, Simon Zhongyuan Tang, Zhonghui Li, Guoliang Ruan, Yijun |
| description | Li
et al
. provide a protocol for long-read ChIA-PET, a technique for mapping chromatin interactions. The longer paired-end tags, which are generated by tagmentation, provide sufficient coverage to determine haplotype-specific chromatin interactions at single-nucleotide resolution.
Chromatin interaction analysis by paired-end tag sequencing (ChIA-PET) is a robust method for capturing genome-wide chromatin interactions. Unlike other 3C-based methods, it includes a chromatin immunoprecipitation (ChIP) step that enriches for interactions mediated by specific target proteins. This unique feature allows ChIA-PET to provide the functional specificity and higher resolution needed to detect chromatin interactions, which chromosome conformation capture (3C)/Hi-C approaches have not achieved. The original ChIA-PET protocol generates short paired-end tags (2 × 20 base pairs (bp)) to detect two genomic loci that are far apart on linear chromosomes but are in spatial proximity in the folded genome. We have improved the original approach by developing long-read ChIA-PET, in which the length of the paired-end tags is increased (up to 2 × 250 bp). The longer PET reads not only improve the tag-mapping efficiency but also increase the probability of covering phased single-nucleotide polymorphisms (SNPs), which allows haplotype-specific chromatin interactions to be identified. Here, we provide the detailed protocol for long-read ChIA-PET that includes cell fixation and lysis, chromatin fragmentation by sonication, ChIP, proximity ligation with a bridge linker, Tn5 tagmentation, PCR amplification and high-throughput sequencing. For a well-trained molecular biologist, it typically takes 6 d from cell harvesting to the completion of library construction, up to a further 36 h for DNA sequencing and |
| doi_str_mv | 10.1038/nprot.2017.012 |
| format | Article |
| fullrecord | <record><control><sourceid>gale_pubme</sourceid><recordid>TN_cdi_pubmedcentral_primary_oai_pubmedcentral_nih_gov_5537732</recordid><sourceformat>XML</sourceformat><sourcesystem>PC</sourcesystem><galeid>A494599336</galeid><sourcerecordid>A494599336</sourcerecordid><originalsourceid>FETCH-LOGICAL-c656t-3f979b17afe89466f53b3e8085180dabc0fe9be4f29414c7851783c7ef9c138b3</originalsourceid><addsrcrecordid>eNqNktFv0zAQxiMEYmPwyiOKxAsIpbPjOLZfJlXVgEoVICji0TjuOfWU2sFOEPvvcbYxVlQQ8oMt3---s---LHuK0Qwjwk9dH_wwKxFmM4TLe9kxZhQVJRPi_tW5KkrMxVH2KMYLhCpGavYwOyo5oZyI6jj7uvKuLQKoTb7YLufFh_N1bnzIGxWh6JUNKRZ9Nw7Wu3yn-t66Nvcm36q-88NlD0XsQVtjda63we_UYF1u3QBB6SknPs4eGNVFeHKzn2SfX5-vF2-L1fs3y8V8Veia1kNBjGCiwUwZ4KKqa0NJQ4AjTjFHG9VoZEA0UJlSVLjSLN0zTjQDIzQmvCEn2dm1bj82O9hocENQneyD3alwKb2ycj_i7Fa2_ruklDBGyiTw4kYg-G8jxEHubNTQdcqBH6NMbcScl4zz_0B5SREmeEKf_4Fe-DG41AlZ0rrGFSUU_4tKZVmalBD1b6pVHUjrjE8f0VNpOa9ERYUgZKJmB6i0NrCz2jswNt3vJbzcS0jMAD-GVo0xyuWnj_vsq7-z8_WXxbuDT9HBxxjA3I4DIznZV17ZV072lcm-KeHZ3SHe4r_8moDTayCmkGsh3GnUYcmf92f4eA</addsrcrecordid><sourcetype>Open Access Repository</sourcetype><iscdi>true</iscdi><recordtype>article</recordtype><pqid>1897394996</pqid></control><display><type>article</type><title>Long-read ChIA-PET for base-pair-resolution mapping of haplotype-specific chromatin interactions</title><source>MEDLINE</source><source>SpringerLink Journals</source><source>Nature Journals Online</source><creator>Li, Xingwang ; Luo, Oscar Junhong ; Wang, Ping ; Zheng, Meizhen ; Wang, Danjuan ; Piecuch, Emaly ; Zhu, Jacqueline Jufen ; Tian, Simon Zhongyuan ; Tang, Zhonghui ; Li, Guoliang ; Ruan, Yijun</creator><creatorcontrib>Li, Xingwang ; Luo, Oscar Junhong ; Wang, Ping ; Zheng, Meizhen ; Wang, Danjuan ; Piecuch, Emaly ; Zhu, Jacqueline Jufen ; Tian, Simon Zhongyuan ; Tang, Zhonghui ; Li, Guoliang ; Ruan, Yijun</creatorcontrib><description>Li
et al
. provide a protocol for long-read ChIA-PET, a technique for mapping chromatin interactions. The longer paired-end tags, which are generated by tagmentation, provide sufficient coverage to determine haplotype-specific chromatin interactions at single-nucleotide resolution.
Chromatin interaction analysis by paired-end tag sequencing (ChIA-PET) is a robust method for capturing genome-wide chromatin interactions. Unlike other 3C-based methods, it includes a chromatin immunoprecipitation (ChIP) step that enriches for interactions mediated by specific target proteins. This unique feature allows ChIA-PET to provide the functional specificity and higher resolution needed to detect chromatin interactions, which chromosome conformation capture (3C)/Hi-C approaches have not achieved. The original ChIA-PET protocol generates short paired-end tags (2 × 20 base pairs (bp)) to detect two genomic loci that are far apart on linear chromosomes but are in spatial proximity in the folded genome. We have improved the original approach by developing long-read ChIA-PET, in which the length of the paired-end tags is increased (up to 2 × 250 bp). The longer PET reads not only improve the tag-mapping efficiency but also increase the probability of covering phased single-nucleotide polymorphisms (SNPs), which allows haplotype-specific chromatin interactions to be identified. Here, we provide the detailed protocol for long-read ChIA-PET that includes cell fixation and lysis, chromatin fragmentation by sonication, ChIP, proximity ligation with a bridge linker, Tn5 tagmentation, PCR amplification and high-throughput sequencing. For a well-trained molecular biologist, it typically takes 6 d from cell harvesting to the completion of library construction, up to a further 36 h for DNA sequencing and <20 h for processing of raw sequencing reads.</description><identifier>ISSN: 1754-2189</identifier><identifier>EISSN: 1750-2799</identifier><identifier>DOI: 10.1038/nprot.2017.012</identifier><identifier>PMID: 28358394</identifier><language>eng</language><publisher>London: Nature Publishing Group UK</publisher><subject>631/337 ; 631/337/100 ; Analytical Chemistry ; Animals ; Biological Techniques ; Chromatin ; Chromatin - metabolism ; Chromatin Immunoprecipitation - methods ; Chromosomes ; Computational Biology/Bioinformatics ; Conformation ; Cytological Techniques - methods ; Deoxyribonucleic acid ; DNA ; DNA - genetics ; DNA - metabolism ; DNA sequencing ; Efficiency ; Gene mapping ; Genomes ; Haplotypes ; Humans ; Immunoprecipitation ; Life Sciences ; Lysis ; Mapping ; Methods ; Microarrays ; Next-generation sequencing ; Nucleotides ; Organic Chemistry ; Physiological aspects ; Positron emission ; Positron emission tomography ; Protein Binding ; Proteins ; Protocol ; RNA polymerase ; Sequence Analysis, DNA - methods ; Single-nucleotide polymorphism ; Sonication ; Tags ; Tomography</subject><ispartof>Nature protocols, 2017-05, Vol.12 (5), p.899-915</ispartof><rights>Springer Nature Limited 2017</rights><rights>COPYRIGHT 2017 Nature Publishing Group</rights><rights>Copyright Nature Publishing Group May 2017</rights><rights>Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved. 2017.</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c656t-3f979b17afe89466f53b3e8085180dabc0fe9be4f29414c7851783c7ef9c138b3</citedby><cites>FETCH-LOGICAL-c656t-3f979b17afe89466f53b3e8085180dabc0fe9be4f29414c7851783c7ef9c138b3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://link.springer.com/content/pdf/10.1038/nprot.2017.012$$EPDF$$P50$$Gspringer$$H</linktopdf><linktohtml>$$Uhttps://link.springer.com/10.1038/nprot.2017.012$$EHTML$$P50$$Gspringer$$H</linktohtml><link.rule.ids>230,314,776,780,881,27901,27902,41464,42533,51294</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/28358394$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Li, Xingwang</creatorcontrib><creatorcontrib>Luo, Oscar Junhong</creatorcontrib><creatorcontrib>Wang, Ping</creatorcontrib><creatorcontrib>Zheng, Meizhen</creatorcontrib><creatorcontrib>Wang, Danjuan</creatorcontrib><creatorcontrib>Piecuch, Emaly</creatorcontrib><creatorcontrib>Zhu, Jacqueline Jufen</creatorcontrib><creatorcontrib>Tian, Simon Zhongyuan</creatorcontrib><creatorcontrib>Tang, Zhonghui</creatorcontrib><creatorcontrib>Li, Guoliang</creatorcontrib><creatorcontrib>Ruan, Yijun</creatorcontrib><title>Long-read ChIA-PET for base-pair-resolution mapping of haplotype-specific chromatin interactions</title><title>Nature protocols</title><addtitle>Nat Protoc</addtitle><addtitle>Nat Protoc</addtitle><description>Li
et al
. provide a protocol for long-read ChIA-PET, a technique for mapping chromatin interactions. The longer paired-end tags, which are generated by tagmentation, provide sufficient coverage to determine haplotype-specific chromatin interactions at single-nucleotide resolution.
Chromatin interaction analysis by paired-end tag sequencing (ChIA-PET) is a robust method for capturing genome-wide chromatin interactions. Unlike other 3C-based methods, it includes a chromatin immunoprecipitation (ChIP) step that enriches for interactions mediated by specific target proteins. This unique feature allows ChIA-PET to provide the functional specificity and higher resolution needed to detect chromatin interactions, which chromosome conformation capture (3C)/Hi-C approaches have not achieved. The original ChIA-PET protocol generates short paired-end tags (2 × 20 base pairs (bp)) to detect two genomic loci that are far apart on linear chromosomes but are in spatial proximity in the folded genome. We have improved the original approach by developing long-read ChIA-PET, in which the length of the paired-end tags is increased (up to 2 × 250 bp). The longer PET reads not only improve the tag-mapping efficiency but also increase the probability of covering phased single-nucleotide polymorphisms (SNPs), which allows haplotype-specific chromatin interactions to be identified. Here, we provide the detailed protocol for long-read ChIA-PET that includes cell fixation and lysis, chromatin fragmentation by sonication, ChIP, proximity ligation with a bridge linker, Tn5 tagmentation, PCR amplification and high-throughput sequencing. For a well-trained molecular biologist, it typically takes 6 d from cell harvesting to the completion of library construction, up to a further 36 h for DNA sequencing and <20 h for processing of raw sequencing reads.</description><subject>631/337</subject><subject>631/337/100</subject><subject>Analytical Chemistry</subject><subject>Animals</subject><subject>Biological Techniques</subject><subject>Chromatin</subject><subject>Chromatin - metabolism</subject><subject>Chromatin Immunoprecipitation - methods</subject><subject>Chromosomes</subject><subject>Computational Biology/Bioinformatics</subject><subject>Conformation</subject><subject>Cytological Techniques - methods</subject><subject>Deoxyribonucleic acid</subject><subject>DNA</subject><subject>DNA - genetics</subject><subject>DNA - metabolism</subject><subject>DNA sequencing</subject><subject>Efficiency</subject><subject>Gene mapping</subject><subject>Genomes</subject><subject>Haplotypes</subject><subject>Humans</subject><subject>Immunoprecipitation</subject><subject>Life Sciences</subject><subject>Lysis</subject><subject>Mapping</subject><subject>Methods</subject><subject>Microarrays</subject><subject>Next-generation sequencing</subject><subject>Nucleotides</subject><subject>Organic Chemistry</subject><subject>Physiological aspects</subject><subject>Positron emission</subject><subject>Positron emission tomography</subject><subject>Protein Binding</subject><subject>Proteins</subject><subject>Protocol</subject><subject>RNA polymerase</subject><subject>Sequence Analysis, DNA - methods</subject><subject>Single-nucleotide polymorphism</subject><subject>Sonication</subject><subject>Tags</subject><subject>Tomography</subject><issn>1754-2189</issn><issn>1750-2799</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2017</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><sourceid>BENPR</sourceid><recordid>eNqNktFv0zAQxiMEYmPwyiOKxAsIpbPjOLZfJlXVgEoVICji0TjuOfWU2sFOEPvvcbYxVlQQ8oMt3---s---LHuK0Qwjwk9dH_wwKxFmM4TLe9kxZhQVJRPi_tW5KkrMxVH2KMYLhCpGavYwOyo5oZyI6jj7uvKuLQKoTb7YLufFh_N1bnzIGxWh6JUNKRZ9Nw7Wu3yn-t66Nvcm36q-88NlD0XsQVtjda63we_UYF1u3QBB6SknPs4eGNVFeHKzn2SfX5-vF2-L1fs3y8V8Veia1kNBjGCiwUwZ4KKqa0NJQ4AjTjFHG9VoZEA0UJlSVLjSLN0zTjQDIzQmvCEn2dm1bj82O9hocENQneyD3alwKb2ycj_i7Fa2_ruklDBGyiTw4kYg-G8jxEHubNTQdcqBH6NMbcScl4zz_0B5SREmeEKf_4Fe-DG41AlZ0rrGFSUU_4tKZVmalBD1b6pVHUjrjE8f0VNpOa9ERYUgZKJmB6i0NrCz2jswNt3vJbzcS0jMAD-GVo0xyuWnj_vsq7-z8_WXxbuDT9HBxxjA3I4DIznZV17ZV072lcm-KeHZ3SHe4r_8moDTayCmkGsh3GnUYcmf92f4eA</recordid><startdate>20170501</startdate><enddate>20170501</enddate><creator>Li, Xingwang</creator><creator>Luo, Oscar Junhong</creator><creator>Wang, Ping</creator><creator>Zheng, Meizhen</creator><creator>Wang, Danjuan</creator><creator>Piecuch, Emaly</creator><creator>Zhu, Jacqueline Jufen</creator><creator>Tian, Simon Zhongyuan</creator><creator>Tang, Zhonghui</creator><creator>Li, Guoliang</creator><creator>Ruan, Yijun</creator><general>Nature Publishing Group UK</general><general>Nature Publishing Group</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>ATWCN</scope><scope>ISR</scope><scope>3V.</scope><scope>7QG</scope><scope>7T5</scope><scope>7T7</scope><scope>7TM</scope><scope>7X7</scope><scope>7XB</scope><scope>88E</scope><scope>8FD</scope><scope>8FE</scope><scope>8FH</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>ABUWG</scope><scope>AEUYN</scope><scope>AFKRA</scope><scope>ATCPS</scope><scope>AZQEC</scope><scope>BBNVY</scope><scope>BENPR</scope><scope>BHPHI</scope><scope>C1K</scope><scope>CCPQU</scope><scope>DWQXO</scope><scope>FR3</scope><scope>FYUFA</scope><scope>GHDGH</scope><scope>GNUQQ</scope><scope>H94</scope><scope>HCIFZ</scope><scope>K9.</scope><scope>LK8</scope><scope>M0S</scope><scope>M1P</scope><scope>M7N</scope><scope>M7P</scope><scope>P64</scope><scope>PATMY</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>PYCSY</scope><scope>RC3</scope><scope>7X8</scope><scope>5PM</scope></search><sort><creationdate>20170501</creationdate><title>Long-read ChIA-PET for base-pair-resolution mapping of haplotype-specific chromatin interactions</title><author>Li, Xingwang ; Luo, Oscar Junhong ; Wang, Ping ; Zheng, Meizhen ; Wang, Danjuan ; Piecuch, Emaly ; Zhu, Jacqueline Jufen ; Tian, Simon Zhongyuan ; Tang, Zhonghui ; Li, Guoliang ; Ruan, Yijun</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c656t-3f979b17afe89466f53b3e8085180dabc0fe9be4f29414c7851783c7ef9c138b3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2017</creationdate><topic>631/337</topic><topic>631/337/100</topic><topic>Analytical Chemistry</topic><topic>Animals</topic><topic>Biological Techniques</topic><topic>Chromatin</topic><topic>Chromatin - metabolism</topic><topic>Chromatin Immunoprecipitation - methods</topic><topic>Chromosomes</topic><topic>Computational Biology/Bioinformatics</topic><topic>Conformation</topic><topic>Cytological Techniques - methods</topic><topic>Deoxyribonucleic acid</topic><topic>DNA</topic><topic>DNA - genetics</topic><topic>DNA - metabolism</topic><topic>DNA sequencing</topic><topic>Efficiency</topic><topic>Gene mapping</topic><topic>Genomes</topic><topic>Haplotypes</topic><topic>Humans</topic><topic>Immunoprecipitation</topic><topic>Life Sciences</topic><topic>Lysis</topic><topic>Mapping</topic><topic>Methods</topic><topic>Microarrays</topic><topic>Next-generation sequencing</topic><topic>Nucleotides</topic><topic>Organic Chemistry</topic><topic>Physiological aspects</topic><topic>Positron emission</topic><topic>Positron emission tomography</topic><topic>Protein Binding</topic><topic>Proteins</topic><topic>Protocol</topic><topic>RNA polymerase</topic><topic>Sequence Analysis, DNA - methods</topic><topic>Single-nucleotide polymorphism</topic><topic>Sonication</topic><topic>Tags</topic><topic>Tomography</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Li, Xingwang</creatorcontrib><creatorcontrib>Luo, Oscar Junhong</creatorcontrib><creatorcontrib>Wang, Ping</creatorcontrib><creatorcontrib>Zheng, Meizhen</creatorcontrib><creatorcontrib>Wang, Danjuan</creatorcontrib><creatorcontrib>Piecuch, Emaly</creatorcontrib><creatorcontrib>Zhu, Jacqueline Jufen</creatorcontrib><creatorcontrib>Tian, Simon Zhongyuan</creatorcontrib><creatorcontrib>Tang, Zhonghui</creatorcontrib><creatorcontrib>Li, Guoliang</creatorcontrib><creatorcontrib>Ruan, Yijun</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Gale In Context: Middle School</collection><collection>Gale In Context: Science</collection><collection>ProQuest Central (Corporate)</collection><collection>Animal Behavior Abstracts</collection><collection>Immunology Abstracts</collection><collection>Industrial and Applied Microbiology Abstracts (Microbiology A)</collection><collection>Nucleic Acids Abstracts</collection><collection>Health & Medical Collection</collection><collection>ProQuest Central (purchase pre-March 2016)</collection><collection>Medical Database (Alumni Edition)</collection><collection>Technology Research Database</collection><collection>ProQuest SciTech Collection</collection><collection>ProQuest Natural Science Collection</collection><collection>Hospital Premium Collection</collection><collection>Hospital Premium Collection (Alumni Edition)</collection><collection>ProQuest Central (Alumni) (purchase pre-March 2016)</collection><collection>ProQuest Central (Alumni Edition)</collection><collection>ProQuest One Sustainability</collection><collection>ProQuest Central UK/Ireland</collection><collection>Agricultural & Environmental Science Collection</collection><collection>ProQuest Central Essentials</collection><collection>Biological Science Collection</collection><collection>ProQuest Central</collection><collection>Natural Science Collection</collection><collection>Environmental Sciences and Pollution Management</collection><collection>ProQuest One Community College</collection><collection>ProQuest Central Korea</collection><collection>Engineering Research Database</collection><collection>Health Research Premium Collection</collection><collection>Health Research Premium Collection (Alumni)</collection><collection>ProQuest Central Student</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>SciTech Premium Collection</collection><collection>ProQuest Health & Medical Complete (Alumni)</collection><collection>ProQuest Biological Science Collection</collection><collection>Health & Medical Collection (Alumni Edition)</collection><collection>Medical Database</collection><collection>Algology Mycology and Protozoology Abstracts (Microbiology C)</collection><collection>Biological Science Database</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>Environmental Science Database</collection><collection>ProQuest One Academic Eastern Edition (DO NOT USE)</collection><collection>ProQuest One Academic</collection><collection>ProQuest One Academic UKI Edition</collection><collection>Environmental Science Collection</collection><collection>Genetics Abstracts</collection><collection>MEDLINE - Academic</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>Nature protocols</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Li, Xingwang</au><au>Luo, Oscar Junhong</au><au>Wang, Ping</au><au>Zheng, Meizhen</au><au>Wang, Danjuan</au><au>Piecuch, Emaly</au><au>Zhu, Jacqueline Jufen</au><au>Tian, Simon Zhongyuan</au><au>Tang, Zhonghui</au><au>Li, Guoliang</au><au>Ruan, Yijun</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Long-read ChIA-PET for base-pair-resolution mapping of haplotype-specific chromatin interactions</atitle><jtitle>Nature protocols</jtitle><stitle>Nat Protoc</stitle><addtitle>Nat Protoc</addtitle><date>2017-05-01</date><risdate>2017</risdate><volume>12</volume><issue>5</issue><spage>899</spage><epage>915</epage><pages>899-915</pages><issn>1754-2189</issn><eissn>1750-2799</eissn><abstract>Li
et al
. provide a protocol for long-read ChIA-PET, a technique for mapping chromatin interactions. The longer paired-end tags, which are generated by tagmentation, provide sufficient coverage to determine haplotype-specific chromatin interactions at single-nucleotide resolution.
Chromatin interaction analysis by paired-end tag sequencing (ChIA-PET) is a robust method for capturing genome-wide chromatin interactions. Unlike other 3C-based methods, it includes a chromatin immunoprecipitation (ChIP) step that enriches for interactions mediated by specific target proteins. This unique feature allows ChIA-PET to provide the functional specificity and higher resolution needed to detect chromatin interactions, which chromosome conformation capture (3C)/Hi-C approaches have not achieved. The original ChIA-PET protocol generates short paired-end tags (2 × 20 base pairs (bp)) to detect two genomic loci that are far apart on linear chromosomes but are in spatial proximity in the folded genome. We have improved the original approach by developing long-read ChIA-PET, in which the length of the paired-end tags is increased (up to 2 × 250 bp). The longer PET reads not only improve the tag-mapping efficiency but also increase the probability of covering phased single-nucleotide polymorphisms (SNPs), which allows haplotype-specific chromatin interactions to be identified. Here, we provide the detailed protocol for long-read ChIA-PET that includes cell fixation and lysis, chromatin fragmentation by sonication, ChIP, proximity ligation with a bridge linker, Tn5 tagmentation, PCR amplification and high-throughput sequencing. For a well-trained molecular biologist, it typically takes 6 d from cell harvesting to the completion of library construction, up to a further 36 h for DNA sequencing and <20 h for processing of raw sequencing reads.</abstract><cop>London</cop><pub>Nature Publishing Group UK</pub><pmid>28358394</pmid><doi>10.1038/nprot.2017.012</doi><tpages>17</tpages><oa>free_for_read</oa></addata></record> |
| fulltext | fulltext |
| identifier | ISSN: 1754-2189 |
| ispartof | Nature protocols, 2017-05, Vol.12 (5), p.899-915 |
| issn | 1754-2189 1750-2799 |
| language | eng |
| recordid | cdi_pubmedcentral_primary_oai_pubmedcentral_nih_gov_5537732 |
| source | MEDLINE; SpringerLink Journals; Nature Journals Online |
| subjects | 631/337 631/337/100 Analytical Chemistry Animals Biological Techniques Chromatin Chromatin - metabolism Chromatin Immunoprecipitation - methods Chromosomes Computational Biology/Bioinformatics Conformation Cytological Techniques - methods Deoxyribonucleic acid DNA DNA - genetics DNA - metabolism DNA sequencing Efficiency Gene mapping Genomes Haplotypes Humans Immunoprecipitation Life Sciences Lysis Mapping Methods Microarrays Next-generation sequencing Nucleotides Organic Chemistry Physiological aspects Positron emission Positron emission tomography Protein Binding Proteins Protocol RNA polymerase Sequence Analysis, DNA - methods Single-nucleotide polymorphism Sonication Tags Tomography |
| title | Long-read ChIA-PET for base-pair-resolution mapping of haplotype-specific chromatin interactions |
| url | https://sfx.bib-bvb.de/sfx_tum?ctx_ver=Z39.88-2004&ctx_enc=info:ofi/enc:UTF-8&ctx_tim=2025-02-06T00%3A05%3A15IST&url_ver=Z39.88-2004&url_ctx_fmt=infofi/fmt:kev:mtx:ctx&rfr_id=info:sid/primo.exlibrisgroup.com:primo3-Article-gale_pubme&rft_val_fmt=info:ofi/fmt:kev:mtx:journal&rft.genre=article&rft.atitle=Long-read%20ChIA-PET%20for%20base-pair-resolution%20mapping%20of%20haplotype-specific%20chromatin%20interactions&rft.jtitle=Nature%20protocols&rft.au=Li,%20Xingwang&rft.date=2017-05-01&rft.volume=12&rft.issue=5&rft.spage=899&rft.epage=915&rft.pages=899-915&rft.issn=1754-2189&rft.eissn=1750-2799&rft_id=info:doi/10.1038/nprot.2017.012&rft_dat=%3Cgale_pubme%3EA494599336%3C/gale_pubme%3E%3Curl%3E%3C/url%3E&disable_directlink=true&sfx.directlink=off&sfx.report_link=0&rft_id=info:oai/&rft_pqid=1897394996&rft_id=info:pmid/28358394&rft_galeid=A494599336&rfr_iscdi=true |