Nanobody-tethered transposition enables multifactorial chromatin profiling at single-cell resolution

Stuart, T.A.; Hao, Stephanie; Zhang, Bingjie; Mekerishvili, Levan; Landau, Dan A.; Maniatis, Silas; Satija, Rahul; Raimondi, Ivan

doi:10.1038/s41587-022-01588-5

Cited by 48 publications

(31 citation statements)

References 45 publications

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“…Profiling of three epigenetic modalities simultaneously has been previously achieved at the bulk level with Multi-CUT&Tag, suggesting that this technology might also allow this trimodal profiling at single-cell level. Here we describe nano-CT, a new multimodal scCUT&Tag protocol, with similarities to NTT-Seq 32 which allows profiling thousands of single cells while (1) reducing the requirements for input material by around 5-fold; (2) increasing the number of fragments detected per cell by 16-fold compared to scCUT&Tag; and (3) allowing the simultaneous probing in each single cell of 3 epigenomic modalities-chromatin accessibility (ATAC-seq), two histone marks (H2K27ac and H3K27me3).…”

Section: Discussionmentioning

confidence: 99%

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Multimodal chromatin profiling using nanobody-based single-cell CUT&Tag

Bartošovič

Castelo‐Branco

2022

Nat Biotechnol

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Probing histone modifications at a single-cell level in thousands of cells has been enabled by technologies such as single-cell CUT&Tag. Here we describe nano-CUT&Tag (nano-CT), which allows simultaneous mapping of up to three epigenomic modalities at single-cell resolution using nanobody-Tn5 fusion proteins. Multimodal nano-CT is compatible with starting materials as low as 25,000–200,000 cells and has significantly higher sensitivity and number of fragments per cell than single-cell CUT&Tag. We use nano-CT to simultaneously profile chromatin accessibility, H3K27ac, and H3K27me3 in juvenile mouse brain, allowing for discrimination of more cell types and states than unimodal single-cell CUT&Tag. We also infer chromatin velocity between assay for transposase-accessible chromatin (ATAC) and H3K27ac in the oligodendrocyte lineage and deconvolute H3K27me3 repressive states, finding two sequential waves of H3K27me3 repression at distinct gene modules during oligodendrocyte lineage progression. Given its high resolution, versatility, and multimodal features, nano-CT allows unique insights in epigenetic landscapes in complex biological systems at the single-cell level.

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Section: Discussionmentioning

confidence: 99%

“…1b,c). This allows profiling of low input bulk samples from as little as ~25,000 cells as starting material, compared to previously required 1 million 16,32 or 150,000 15 cells for scCUT&Tag protocols on the Chromium platform.…”

Section: Unimodal Single-cell Nano-ct Outperforms Sccutandtagmentioning

confidence: 99%

Multimodal chromatin profiling using nanobody-based single-cell CUT&Tag

Bartošovič

Castelo‐Branco

2022

Nat Biotechnol

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“…We anticipate that future studies will extend Phospho-seq to capture additional modalities related to chromatin state, and may shed additional light towards our understanding of how transcription factors regulate cellular chromatin. For example, combining Phospho-seq with scCUT&Tag 58 or NTT-seq 59 would identify TFs whose abundance correlated not only with chromatin accessibility, but with the presence of either activating or repressive chromatin marks. Further extensions that enable guide RNA capture (i.e.…”

Section: Discussionmentioning

confidence: 99%

Phospho-seq: Integrated, multi-modal profiling of intracellular protein dynamics in single cells

Blair

Hartman

Zenk

et al. 2023

Preprint

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Cell signaling plays a critical role in regulating cellular behavior and fate. While multimodal single-cell sequencing technologies are rapidly advancing, scalable and flexible profiling of cell signaling states alongside other molecular modalities remains challenging. Here we present Phospho-seq, an integrated approach that aims to quantify phosphorylated intracellular and intranuclear proteins, and to connect their activity with cis-regulatory elements and transcriptional targets. We utilize a simplified benchtop antibody conjugation method to create large custom antibody panels for simultaneous protein and scATAC-seq profiling on whole cells, and integrate this information with scRNA-seq datasets via bridge integration. We apply our workflow to cell lines, induced pluripotent stem cells, and 3-month-old brain organoids to demonstrate its broad applicability. We demonstrate that Phospho-seq can define cellular states and trajectories, reconstruct gene regulatory relationships, and characterize the causes and consequences of heterogeneous cell signaling in neurodevelopment.

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“…A common challenge for all the current methods simultaneously profiling epigenetic modifications is the low coverage obtained from single cells 21,23,24,26 , which is also true for MAbID single-cell data. Even though combined profiling inherently creates a rich dataset, the sparsity of reads hampers studying the relationship between epitopes, for example when investigating co-occupancy.…”

Section: Discussionmentioning

confidence: 99%

Combinatorial single-cell profiling of all major chromatin types with MAbID

Lochs

Weide

Luca

et al. 2023

Preprint

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Gene expression programs result from the collective activity of many regulatory factors. To obtain insight into the mechanisms that govern gene regulation, it is imperative to study their combined mode of action and interconnectivity. However, it has been challenging to simultaneously measure a combination of these factors within one sample. Here, we introduce MAbID, a method that combines genomic profiling of many histone modifications and chromatin-binding proteins in a single reaction. MAbID employs antibody-DNA conjugates to enable genomic barcoding of chromatin at sites of epitope occupancy. This barcoding strategy allows for the combined incubation of multiple antibodies in a single sample to reveal the genomic distributions of many epigenetic states simultaneously. We used MAbID to profile both active and inactive chromatin types in human cell lines and multiplexed measurements in the same sample without loss of data quality. Moreover, we obtained joint measurements of six epitopes covering all major chromatin types in single cells during mouse in vitro neural differentiation and captured associated changes in multifactorial chromatin states. Thus, MAbID holds the potential to gain unique insights into the interplay between gene regulatory mechanisms, especially in settings with limited sample material and in single cells.

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Nanobody-tethered transposition enables multifactorial chromatin profiling at single-cell resolution

Cited by 48 publications

References 45 publications

Multimodal chromatin profiling using nanobody-based single-cell CUT&Tag

Multimodal chromatin profiling using nanobody-based single-cell CUT&Tag

Phospho-seq: Integrated, multi-modal profiling of intracellular protein dynamics in single cells

Combinatorial single-cell profiling of all major chromatin types with MAbID

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