One Sentence Summary:A new method, CHEX-seq (CHromatin eXposed), identifies the openchromatin landscape in single fixed cells thereby allowing spatial chromatin analysis of selected cells in complex cellular environments.
AbstractAssays examining the open-chromatin landscape in single cells require isolation of the nucleus, resulting in the loss of spatial/microenvironment information. Here we describe CHEX-seq (CHromatin EXposed) for identifying single-stranded open-chromatin DNA regions in paraformaldehyde-fixed single cells. CHEX-seq uses light-activated DNA probes that binds to single-stranded DNA in open chromatin.In situ laser activation of the annealed probes' 3'-Lightning Terminator™ in selected cells permits the probe to act as a primer for in situ enzymatic copying of single-stranded DNA that is then sequenced.CHEX-seq is benchmarked with human K562 cells and its utility is demonstrated in dispersed primary mouse and human brain cells, and immunostained cells in mouse brain sections. Further, CHEX-seq queries the openness of mitochondrial DNA in single cells. Evaluation of an individual cell's chromatin landscape in its tissue context enables "spatial chromatin analysis".
Main TextThe process of RNA transcription requires a cell's genomic DNA to be in an open-chromatin conformation, where there is less nucleosome packing, so that the transcription regulatory proteins can bind and function. It is clear that chromatin structure is dynamic and regulated by a number of factors including development, stress, and pharmacological challenge (1-3). Most chromatin modeling studies have relied upon the use of pooled cells to generate genomic DNA/chromatin for analysis. Included among chromatin analysis procedures are DNase-seq, FAIRE-seq, and ChIP-seq as well as other approaches. Recently, these methods have been extended to single cells (4-7). For example, the recent ATAC-seq approach to mapping chromatin in single cells exploits an assay for detecting transposon-accessible chromatin (8). This methodology uses Tn5 transposase to tag and purify accessible nucleosome-free double-stranded DNA regions in the genome. Each of these procedures has specific advantages and disadvantages, with the most significant disadvantage being that they all assess chromatin in nuclei isolated from the tissue of interest, thereby losing spatial location information and the cellular microenvironment context. To overcome this issue, we developed CHEXseq (CHromatin EXposed) to assess chromatin conformation in fixed single cells including neurons and astrocytes.