Multifactorial profiling of epigenetic landscapes at single-cell resolution using MulTI-Tag

Abstract

Chromatin profiling at locus resolution uncovers gene regulatory features that define cell types and developmental trajectories, but it remains challenging to map and compare different chromatin-associated proteins in the same sample. Here we describe Multiple Target Identification by Tagmentation (MulTI-Tag), an antibody barcoding approach for profiling multiple chromatin features simultaneously in single cells. We optimized MulTI-Tag to retain high sensitivity and specificity, and we demonstrate detection of up to three histone modifications in the same cell: H3K27me3, H3K4me1/2 and H3K36me3. We apply MulTI-Tag to resolve distinct cell types and developmental trajectories; to distinguish unique, coordinated patterns of active and repressive element regulatory usage associated with differentiation outcomes; and to uncover associations between histone marks. Multifactorial epigenetic profiling holds promise for comprehensively characterizing cell-specific gene regulatory landscapes in development and disease.

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

“…Together, these analyses demonstrate that NTT-seq datasets provide accurate multimodal chromatin landscapes at single-cell resolution; contain sufficient information to identify major cell types and states in primary human tissues; provide profiles that reflect high-quality bulk ChIP-seq data 17 ; and can be generated in conjunction with accurate cell surface protein expression measurements. Existing multimodal chromatin technologies require complex experimental workflows and have not been demonstrated to work with complex tissue samples 6,7 or are strictly limited in the chromatin states that they can measure 23 . NTT-seq overcomes both of these limitations, providing a streamlined experimental workflow applicable to complex tissues.…”

“…We first created a salmon index 35 for the BioLegend TotalSeq-A antibody panel, with the -features -k7 parameters. We quantified counts for each ADT barcode using the salmon Alevin command with the following parameters: -naiveEqclass, -keepCBFraction 0.8, -bc-geometry 1 [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16], -umi-geometry 2[1-10], -read-geometry 2[71-85].…”

“…Most single-cell chromatin profiling methods employ protein-A/G fused to Tn5 transposase [1][2][3]6,7 . Protein-A/G binds to IgG antibodies, enabling Tn5 to be directed to regions of the genome where an IgG antibody is bound and inserting adapters for DNA sequencing.…”

“…As protein-A/G binds to IgG antibodies from different species with high affinity, such methods are difficult to perform in an antibody-multiplexed design aiming to measure multiple histone modifications in a single experiment. Current approaches for multimodal chromatin profiling using protein-A/G, such as MulTI-Tag, involve complex experimental workflows with multiple wash and incubation steps 7 . Such methods have not been demonstrated to work with complex tissues 6,7 , thus limiting their broader application.…”

“…Current approaches for multimodal chromatin profiling using protein-A/G, such as MulTI-Tag, involve complex experimental workflows with multiple wash and incubation steps 7 . Such methods have not been demonstrated to work with complex tissues 6,7 , thus limiting their broader application. We reasoned that the use of small single polypeptide chain antibodies (nanobodies) that specifically bind IgG from different species or different IgG subtypes in place of protein-A/G may enable the multiplexing of primary antibodies to facilitate a multimodal chromatin assay 8 .…”