Non-destructive handling of individual chromatin fibers isolated from single cells in a microfluidic device utilizing an optically driven microtool

Abstract

We report a novel method for the non-destructive handling of, and biochemical experiments with, individual intact chromatin fibers, as well as their isolation from single cells, utilizing a specifically designed microfluidic device with an optically driven microtool under the microscope. Spheroplasts of recombinant fission yeast cells expressing fluorescent protein-tagged core histones were employed, and isolation of chromatin fibers was conducted by cell bursting via changing from isotonic conditions to hypotonic conditions in the microfluidic device. The isolation of chromatin fibers was confirmed by the fluorescent protein-tagged core histones involved in the chromatin fibers. For the non-destructive handling of the isolated chromatin fibers in the microfluidic device, we developed antibody-conjugated microspheres, which had affinity to the fluorescent protein-tagged core histones, and the microspheres were manipulated using optical tweezers, which functioned as optically driven microtools. With the aid of the microtool, isolated chromatin fibers were handled non-destructively and were tethered at the microstructures fabricated in the microfluidic device with straightened conformation by the flow. Immunofluorescence staining was carried out as a demonstrative biochemical experiment with the individual native chromatin fibers isolated in the microfluidic device, and specific fluorescent spots were visualized along the tethered chromatin fibers. Thus, the potential application of this method for epigenetic analyses of intact chromatin fibers isolated from single cells is demonstrated.

Nucleosomes Exhibit Non‐uniform Unwrapping Along Native Chromatin Fibers with Increasing Salt Concentration as Revealed by Direct Imaging in a Microfluidic Channel

“…The main channel (height of ≈20 μm) had micropockets and micropillars, with the former being used as reaction chambers in which cells and chromatin fibers were confined during solution exchange owing to their small diffusion coefficients and isolation from streamlines in the main channel. We previously demonstrated that solution conditions within the chambers could be rapidly altered by diffusion . For instance, chromatin fibers were isolated without fragmentation from cells when a hypotonic solution was introduced into the main channel .…”

“…We previously demonstrated that solution conditions within the chambers could be rapidly altered by diffusion . For instance, chromatin fibers were isolated without fragmentation from cells when a hypotonic solution was introduced into the main channel . The micropillars were used to immobilize and tether isolated chromatin fibers.…”

“…We recently developed a novel method for non‐destructive handling of individual chromatin fibers isolated from single cells in a microfluidic channel under a fluorescence microscope . Isolated chromatin fibers were tethered and elongated by flow and changes in higher‐order structure were induced by replacing the solution in the microfluidic channel.…”

Single molecule and single cell epigenomics

“…The C1 system from Fluidigm facilitates isolation of single cells, as well as on-device extraction and processing of RNA and DNA for such studies; however, this platform has not been adapted for chromatin isolation. In an effort to fill this gap, microscale devices have been developed capable of trapping low inputs of cells for standard ChIP [41, 118] and restriction enzyme based 5mC analysis [119], from single cells that enable the isolation and manipulation of single chromatin molecules using antibody conjugated microspheres and optical tweezers [120], or from single cells using a design that enables their integration with the SCAN platform [121]. The latter has been used for extracting chromatin from low cell inputs by sequentially flowing into the devices the reagents used for native chromatin extractions, followed by fluorescent antibodies needed for detecting chromatin features by SCAN (unpublished).…”

Single-Cell Sequencing for Precise Cancer Research: Progress and Prospects

“…This method filled the gaps between genomics and microscopy analyses of chromosomes, enabling the study of gene regulation of cancer based on chromatin organization. A few other tools for epigenetic analyses of single-cell chromatin have also been reported, such as the single chromatin molecule analysis at the nanoscale (SCAN) platform (62), and methods using antibody-based individual intact chromatin fiber labeling, capture and image analysis (63).…”