Abstract
Cleavage under targets and tagmentation (CUT&Tag) is an antibody-directed in situ chromatin profiling strategy that is rapidly replacing immune precipitation-based methods, such as chromatin immunoprecipitation–sequencing. The efficiency of the method enables chromatin profiling in single cells but is limited by the numbers of cells that can be profiled. Here, we describe a combinatorial barcoding strategy for CUT&Tag that harnesses a nanowell dispenser for simple, high-resolution, high-throughput, single-cell chromatin profiling. In this single-cell combinatorial indexing CUT&Tag (sciCUT&Tag) protocol, lightly cross-linked nuclei are bound to magnetic beads and incubated with primary and secondary antibodies in bulk and then arrayed in a 96-well plate for a first round of cellular indexing by antibody-directed Tn5 tagmentation. The sample is then repooled, mixed and arrayed across 5,184 nanowells at a density of 12–24 nuclei per well for a second round of cellular indexing during PCR amplification of the sequencing-ready library. This protocol can be completed in 1.5 days by a research technician, and we illustrate the optimized protocol by profiling histone modifications associated with developmental gene repression (H3K27me3) as well as transcriptional activation (H3K4me1-2-3) in human peripheral blood mononuclear cells and use single-nucleotide polymorphisms to facilitate collision removal. We have also used sciCUT&Tag for simultaneous profiling of multiple chromatin epitopes in single cells. The reduced cost, improved resolution and scalability of sciCUT&Tag make it an attractive platform to profile chromatin features in single cells.
Key points
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This protocol describes a method for the single-cell profiling of epigenetic marks.
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Compared with other single-cell chromatin profiling methods, single-cell combinatorial indexing cleavage under targets and tagmentation can profile a greater number of cells while maintaining the optimal reaction chemistry for efficient PCR amplification of tagmented chromatin, thereby increasing the number of unique reads recovered per cell relative to droplet-based approaches.
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Data availability
The primary sequencing data from the barnyard experiment, mixing human and mouse cell lines, as well as single-donor and mixed-donor PBMC sciCUT&Tag experiments have been deposited as paired-end fastq files that retain the unique cellular barcodes in the Gene Expression Omnibus accession GSE224579.
Code availability
Custom code used for demultiplexing, concatenating and aligning to hg38 and mm10 reference genomes for the barnyard experiment (Snakemake41), and running Souporcell24 (Nextflow26) as well as code for generating figures is available at https://github.com/FredHutch/sciCnT_manuscript_2023. Processed data used for making figures is available at https://doi.org/10.5281/zenodo.7884460. Aggregated sciCnT PBMC coverage profiles and peak calls are interactively available at https://genome.ucsc.edu/s/jegreene/sciCnT_HOX.
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Acknowledgements
We thank members of our laboratory and colleagues at the Fred Hutch for providing valuable discussions and input as we developed this protocol. Specifically, we thank Fred Hutchinson Cancer Center Genomics and Bioinformatics Core Facility for sequencing expertise and services; M. Fitzgibbons for help with demultiplexing, J. Henikoff for help with genomic alignment; M. Setty, D.Otto, A.-L. Doebley and O. Waltner for bioinformatics advice; S. Bhise for help with preparing the human PBMC samples; and J. Cherone, P. Zrazhevskiy and the Andrew Stergachis laboratory for their helpful feedback on the manuscript. We are especially grateful to the many Protocols.io subscribers around the world who have tried CUT&Tag and provided helpful comments and feedback that have enriched this protocol. This work was supported by the Howard Hughes Medical Institute (S.H.), grant R01 HG010492 (S.H.) from the National Institutes of Health, and a Hartwell Foundation Postdoctoral Fellowship (D.H.J.).
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Authors and Affiliations
Contributions
D.H.J., J.E.G., S.J.W., K.A. and S.H. conceived the study. J.E.G., D.H.J., S.J.W. and C.A.C. optimized the sciCUT&Tag protocol on the ICELL8. J.E.G. and D.H.J. designed the experiments and carried them out with the help of C.A.C. D.H.J. and J.E.G. performed the data analysis and generated the figures. S.N.F. provided the human PBMC samples, advised on the implementation of Souporcell for doublet detection and provided helpful discussion. S.S.M. constructed an easy-to-use Nextflow pipeline for running Souporcell. J.E.G., D.H.J., C.A.C., K.A. and S.H. wrote the manuscript. All authors read and approved the final manuscript.
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S.H. has filed patent applications on related work. The other authors declare no competing interests.
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Nature Protocols thanks Julia Zeitlinger and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
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Related links
Key references using this protocol
Kaya-Okur, H. S. et al. Nat. Commun. 10, 1930 (2019): https://doi.org/10.1038/s41467-019-09982-5
Wu, S. J. et al. Nat. Biotechnol. 39, 819–824 (2021): https://doi.org/10.1038/s41587-021-00865-z
Meers, M. P. et al. Nat. Biotechnol. 41, 708–716 (2023): https://doi.org/10.1038/s41587-022-01522-9
Supplementary information
Supplementary Information
Supplementary Methods and Figs. 1 and 2.
Supplementary Table 1
All the barcoded Tn5 s5 and s7 and universal MErev adapter sequences are shown in the first tab, the barcoded i5 and i7 primer sequences in the second tab and the custom primer sequences used for illumine sequencing in the third tab.
Supplementary Table 2
Layout of the 96-well sci-pA–Tn5 plate
Supplementary Table 3
Layout of the 384-well i5 and i7 primer plate for dispensing on the ICell8 instrument
Supplementary Table 4
An example of the input file for the sciCTextract custom software that assigns sequencing reads to sample name prefixes based on the s5 and s7 adapter sequence
Supplementary Table 5
An example of the input file for the sciCTextract custom software that assigns sequencing reads to sample name suffixes based on the i5 and i7 adapter sequence
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Janssens, D.H., Greene, J.E., Wu, S.J. et al. Scalable single-cell profiling of chromatin modifications with sciCUT&Tag. Nat Protoc 19, 83–112 (2024). https://doi.org/10.1038/s41596-023-00905-9
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DOI: https://doi.org/10.1038/s41596-023-00905-9
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