The red line indicates average enrichment for all those single cells from your iACT-seq data set. cells. ACT-seq utilizes a fusion of Tn5 transposase to Protein A that is targeted to chromatin by a specific antibody, allowing chromatin fragmentation and sequence tag insertion specifically at genomic sites presenting the relevant antigen. The Tn5 transposase enables the use of an index multiplexing strategy (iACT-seq), which enables construction of thousands of single-cell libraries in one day by a single researcher without the need for drop-based fluidics or visual sorting. We conclude that ACT-seq present a stylish alternative to existing techniques for mapping epigenetic marks in single cells. Subject terms: Genomic analysis, Histone post-translational Rabbit Polyclonal to SLC25A12 modifications, Histone variants, Epigenetics The CRAC intermediate 2 authors expose ACT-seq: a Tn5-based method for rapidly profiling epigenetic marks in bulk-cell and single-cell samples. ACT-seq avoids many laborious or time-consuming actions required for comparable techniques including chromatin fragmentation and end repair. Introduction Techniques for mapping epigenetic says in individual cells have enhanced our understanding of differentiation and cell-to-cell variance. Multiple single-cell methods have been developed to map transcriptomes and chromatin business, and these methods are proving priceless in fields such as cancer research1,2. Comparatively few methods exist for single-cell profiling of histone tail modifications1,3, which play important functions in epigenetic control of gene expression and development4. Here we describe antibody-guided chromatin tagmentation sequencing (ACT-seq), a technique to assay distributions of epigenetic marks in a small number of cells or thousands of single cells simultaneously. ACT-seq utilizes Tn5 transposase, which is commonly used to map chromatin convenience and structure5C7. We fused the N terminus of Tn5 transposase to Protein A (PA) to form a fusion protein hereafter referred to as PA-Tnp (Supplementary Fig.?1). The PA domain name of the fusion protein is first bound to an antibody that is selected to target an epigenetic mark or chromatin-bound protein of interest. The complex is usually then incubated with permeabilized cells and is guided to chromatin by the associated antibody. After washing away unbound complex, the transposition reaction is initiated by addition of an MgCl2-made up of buffer, which results in insertion of sequence tags at sites of bound PA-Tnp. The reaction is usually terminated by incubation with EDTA and proteinase. The labeled fragments are directly amplified CRAC intermediate 2 using PCR and sequenced using Illumina HiSeq technology. Results ACT-seq robustly maps epigenetic marks in bulk-cell samples To evaluate the efficiency of ACT-seq, we mapped the distributions of a variety of epigenetic features in HEK293T cells: the histone tail modifications H3K4me1, H3K4me2, H3K4me3, and H2K27ac; the histone variant H2A.Z; and the chromatin-binding protein Brd4. Visual inspection using a genome browser revealed highly comparable distributions of enrichment in the bulk-cell ACT-seq data relative to published ChIP-seq data sets (Fig.?1a)8,9. By comparison, little to no specific enrichment was apparent in the ACT-seq mock IgG sample, indicating that the observed signals were antibody-specific. Analysis of statistically significant peaks of enrichment also revealed strong correlations between the data sets obtained using ACT-seq and ChIP-seq (Supplementary Fig.?2). Further, we detected strong average enrichment of H3K27ac, H2A.Z, and Brd4 at transcription start sites (TSS) and enhancer regions in our ACT-seq data (Fig.?1b, c) in agreement with published studies on these factors10C12. To determine whether the ACT-seq transmission was distinguishable from nonspecific Tn5 activity at regions of open chromatin, we statistically compared the H3K4me3 ACT-seq peaks with a published ATAC-seq data set that was generated using the same cell type13. We found that over 11,000 ACT-seq peaks remained after CRAC intermediate 2 filtering using a false discovery rate threshold of 0.05, indicating that ACT-seq peaks predominantly arise from antibody binding as opposed to nonspecific Tn5 activity (Supplementary Fig.?3). Taken together, these analyses confirm that ACT-seq and ChIP-seq provide comparable information on enrichment of epigenetic marks in bulk-cell samples. Open in a separate windows Fig. 1 ACT-seq robustly maps epigenetic marks in bulk-cell samples. a Genome browser image.