title: Chromatin in situ proximity (ChrISP): single-cell analysis of chromatin proximities at a high resolution.
abstract: Current techniques for analyzing chromatin structures are hampered by either poor resolution at the individual cell level or the need for a large number of cells to obtain higher resolution. This is a major problem as it hampers our understanding of chromatin conformation in single cells and how these respond to environmental cues. Here we describe a new method, chromatin in situ proximity (ChrISP), which reproducibly scores for proximities between two different chromatin fibers in 3-D with a resolution of ~170Å in single cells. The technique is based on the in situ proximity ligation assay (ISPLA), but ChrISP omits the rolling circle amplification step (RCA). Instead, the proximities between chromatin fibers are visualized by a fluorescent connector oligonucleotide DNA, here termed splinter, forming a circular DNA with another circle-forming oligonucleotide, here termed backbone, upon ligation. In contrast to the regular ISPLA technique, our modification enables detection of chromatin fiber proximities independent of steric hindrances from nuclear structures. We use this method to identify higher order structures of individual chromosomes in relation to structural hallmarks of interphase nuclei and beyond the resolution of the light microscope.
authors.affiliation: Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Xingqi
authors.initials: X
authors.lastname: Chen
authors.affiliation: Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Chengxi
authors.initials: C
authors.lastname: Shi
authors.affiliation: Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Samer
authors.initials: S
authors.lastname: Yammine
authors.affiliation: Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Anita
authors.initials: A
authors.lastname: Göndör
authors.affiliation: Applied Physics, AlbaNova University Center, Royal Institute of Technology, Sweden.
authors.forename: Daniel
authors.initials: D
authors.lastname: Rönnlund
authors.affiliation: Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Alejandro
authors.initials: A
authors.lastname: Fernandez-Woodbridge
authors.affiliation: Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Noriyuki
authors.initials: N
authors.lastname: Sumida
authors.affiliation: Applied Physics, AlbaNova University Center, Royal Institute of Technology, Sweden.
authors.forename: Jerker
authors.initials: J
authors.lastname: Widengren
authors.affiliation: Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Rolf
authors.initials: R
authors.lastname: Ohlsson
mesh_terms: D002843:Chromatin / Q000737:chemistry*; D002880:Chromosomes, Human, Pair 11; D004270:DNA, Circular; D045325:HCT116 Cells; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods; D008853:Microscopy / Q000379:methods*; D009690:Nucleic Acid Conformation; D009693:Nucleic Acid Hybridization / Q000379:methods*; D011487:Protein Conformation; D012680:Sensitivity and Specificity; D059010:Single-Cell Analysis
chemical_list: D002843:Chromatin; D004270:DNA, Circular
keywords: chromosome; conformation; epigenetics