Chromatin integration labeling for mapping dna-binding proteins and modifications with low input

ABSTRACT Cell identity is determined by the selective activation or silencing of specific genes via transcription factor binding and epigenetic modifications on the genome. Chromatin

immunoprecipitation (ChIP) has been the standard technique for mapping the sites of transcription factor binding and histone modification. Recently, alternative methods to ChIP have been

developed for addressing the increasing demands for low-input epigenomic profiling. Chromatin integration labeling (ChIL) followed by sequencing (ChIL-seq) has been demonstrated to be

particularly useful for epigenomic profiling of low-input samples or even single cells because the technique amplifies the target genomic sequence before cell lysis. After labeling the

target protein or modification in situ with an oligonucleotide-conjugated antibody (ChIL probe), the nearby genome sequence is amplified by Tn5 transposase-mediated transposition followed by

T7 RNA polymerase-mediated transcription. ChIL-seq enables the detection of the antibody target localization under a fluorescence microscope and at the genomic level. Here we describe the

detailed protocol of ChIL-seq with assessment methods for the key steps, including ChIL probe reaction, transposition, in situ transcription and sequencing library preparation. The protocol

usually takes 3 d to prepare the sequencing library, including overnight incubations for the ChIL probe reaction and in situ transcription. The ChIL probe can be separately prepared and

stored for several months, and its preparation and evaluation protocols are also documented in detail. An optional analysis for multiple targets (multitarget ChIL-seq) is also described. We

anticipate that the protocol presented here will make the ChIL technique more widely accessible for analyzing precious samples and facilitate further applications. Access through your

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CHROMATIN IMMUNOPRECIPITATION SEQUENCING FOR QUANTITATIVE STUDY OF HISTONE MODIFICATIONS AND CHROMATIN FACTORS Article 03 October 2024 SCNANOSEQ-CUT&TAG: A SINGLE-CELL LONG-READ

CUT&TAG SEQUENCING METHOD FOR EFFICIENT CHROMATIN MODIFICATION PROFILING WITHIN INDIVIDUAL CELLS Article 07 October 2024 EFFICIENT LOW-COST CHROMATIN PROFILING WITH CUT&TAG Article

10 September 2020 DATA AVAILABILITY ChIL-seq and mtChIL-seq data in this study have been deposited in the Gene Expression Omnibus (GEO) under the accession code GSE140659. Source data are

provided with this paper. CODE AVAILABILITY All performance metrics of low-input epigenomic profiling methods in Table 2 are the re-evaluation of our original ChIL-seq data analysis (except

for the adapted information from previous reports, specified in the table legend). The main workhorse is the tableTPFP function in RScripts/myROC.R in

https://github.com/kazumits/DataAnalysisForChILT. The use case of calculating ROC at TSS is according to the example described at

https://github.com/kazumits/DataAnalysisForChILT/blob/master/ChILT_optimalResolution.md. The evaluation of genome-wide prediction performance (for example, precision, recall) against

gold-standard ChIP-seq peaks is shown at https://github.com/kazumits/DataAnalysisForChILT/blob/master/ChILT_sensitivity-specificity.md. Source data are provided with this paper. CHANGE

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references ACKNOWLEDGEMENTS We are grateful to the staff of Ohkawa Lab. The computation was carried out using the computer resource offered under the category of Intensively Promoted

Projects by Research Institute for Information Technology, Kyushu University. This work was in part supported by MEXT/JSPS KAKENHI (JP17K17719 to T.H.; JP18K19432, JP19H03211, JP19H05425 and

JP20H05368 to A.H.; JP18H04904, JP19H04970, JP19H03158 and JP20H05393 to K.M.; JP18H05534 to H.Ku.; JP18H04802, JP18H05527, JP19H05244, JP17H03608, JP20H00456, JP20H04846 and JP20K21398 to

Y.O.; and JP18H05527 and JP17H01417 to H.Ki), JST CREST (JPMJCR16G1 to Y.O., H.Ku. and H.Ki.), JST PRESTO JPMJPR19K7 to A.H, AMED JP20ek0109489h0001 to Y.O. and AMED BINDS (JP19am0101076 to

H.Ku. and JP19am0101105 to H.Ki.). AUTHOR INFORMATION Author notes * These authors contributed equally: Tetsuya Handa, Akihito Harada, Kazumitsu Maehara. AUTHORS AND AFFILIATIONS * Cell

Biology Center, Institute of Innovative Research, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama, Japan Tetsuya Handa & Hiroshi Kimura * Division of Transcriptomics,

Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, Japan Akihito Harada, Kazumitsu Maehara & Yasuyuki Ohkawa * Institute for Quantitative

Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, Japan Shoko Sato & Hitoshi Kurumizaka * Graduate School of Bioscience and Biotechnology, Tokyo Institute of

Technology, 4259 Nagatsuta, Midori-ku, Yokohama, Japan Masaru Nakao, Naoki Goto & Hiroshi Kimura Authors * Tetsuya Handa View author publications You can also search for this author

inPubMed Google Scholar * Akihito Harada View author publications You can also search for this author inPubMed Google Scholar * Kazumitsu Maehara View author publications You can also search

for this author inPubMed Google Scholar * Shoko Sato View author publications You can also search for this author inPubMed Google Scholar * Masaru Nakao View author publications You can

also search for this author inPubMed Google Scholar * Naoki Goto View author publications You can also search for this author inPubMed Google Scholar * Hitoshi Kurumizaka View author

publications You can also search for this author inPubMed Google Scholar * Yasuyuki Ohkawa View author publications You can also search for this author inPubMed Google Scholar * Hiroshi

Kimura View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS T.H., A.H., Y.O. and H. Ki. designed the experiments. T.H. and A.H. performed the

ChIL experiments with deep-sequencing analyses. K.M. performed the bioinformatics and statistical analyses. S.S. and H.Ku. produced the in-house Tn5 transposase. M.N. and N.G. contributed to

the assay for in situ transcription. T.H., A.H., K.M., Y.O. and H.Ki. wrote the manuscript. All authors read and approved the final manuscript. CORRESPONDING AUTHORS Correspondence to

Yasuyuki Ohkawa or Hiroshi Kimura. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing financial interests except T.H., A.H., H. Ku., Y.O. and H.Ki, who are involved in

a pending patent related to ChIL. ADDITIONAL INFORMATION PEER REVIEW INFORMATION _Nature Protocols_ thanks Chang Lu, Sinem K. Saka and the other, anonymous, reviewer(s) for their

contribution to the peer review of this work. PUBLISHER’S NOTE Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. RELATED

LINKS KEY REFERENCE USING THIS PROTOCOL: Harada, A. et al. _Nat. Cell Biol_. 21, 287−296 (2019): https://doi.org/10.1038/s41556-018-0248-3 SUPPLEMENTARY INFORMATION SUPPLEMENTARY INFORMATION

Supplementary Tables 1–5 and Figs. 1–9. REPORTING SUMMARY SOURCE DATA SOURCE DATA FIG. 2 Unprocessed gels. SOURCE DATA FIG. 3 Statistical source data. SOURCE DATA FIG. 4 Statistical source

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permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Handa, T., Harada, A., Maehara, K. _et al._ Chromatin integration labeling for mapping DNA-binding proteins and modifications with low input.

_Nat Protoc_ 15, 3334–3360 (2020). https://doi.org/10.1038/s41596-020-0375-8 Download citation * Received: 24 November 2019 * Accepted: 29 May 2020 * Published: 17 August 2020 * Issue Date:

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