Chromatin immunoprecipitation in microfluidic droplets: towards fast and cheap analyses

Abstract

Genetic organization is governed by the interaction of DNA with histone proteins, and differential modifications of these proteins is a fundamental mechanism of gene regulation. Histone modifications are primarily studied through chromatin immunoprecipitation (ChIP) assays, however conventional ChIP procedures are time consuming, laborious and require a large number of cells. Here we report for the first time the development of ChIP in droplets based on a microfluidic platform combining nanoliter droplets, magnetic beads (MB) and magnetic tweezers (MT). The droplet approach enabled compartmentalization and improved mixing, while reducing the consumption of samples and reagents in an integrated workflow. Anti-histone antibodies grafted to MB were used as a solid support to capture and transfer the target chromatin from droplets to droplets in order to perform chromatin immunoprecipitation, washing, elution and purification of DNA. We designed a new ChIP protocol to investigate four different types of modified histones with known roles in gene activation or repression. We evaluated the performances of this new ChIP in droplet assay in comparison with conventional methods. The proposed technology dramatically reduces analytical time from a few days to 7 hours, simplifies the ChIP protocol and decreases the number of cells required by 100 fold while maintaining a high degree of sensitivity and specificity. Therefore this droplet-based ChIP assay represents a new, highly advantageous and convenient approach to epigenetic analyses.

High-Throughput Incubation and Quantification of Agglutination Assays in a Microfluidic System

“…We routinely measured negative test assays run through the system and found no detectable cross-contamination between consecutive assays as expected. This characteristic is consistent with the literature, as some researchers [ 63 , 77 ] have shown similar plug-based assays in which microbeads are magnetically manipulated and transported from one aqueous plug to another, with no signs of cross-contamination.…”

Perspectives on the Role of Histone Modification in Breast Cancer Progression and the Advanced Technological Tools to Study Epigenetic Determinants of Metastasis

“…Moreover, the ultrasonic sonication was also recently integrated into the microfluidic chip in order to facilitate efficient mixing and washing in the ChIP process (Cao and Lu, 2016). A droplet-based microfluidic chip was reported to reduce cross-contamination and enhanced mixing in the nanoliter scale (Teste et al, 2017). The droplet microfluidic device can also optimize the fragmentation of chromatin process to improve the efficiency of downstream epigenomic assays, such as ChIP (Xu et al, 2018).…”

Droplet CAR-Wash: continuous picoliter-scale immunocapture and washing

“…We chose a HeLa cell line expressing a green fluorescent protein-histone H2B (GFP-H2B) fusion in the nucleus to provide a target protein to fluorescently monitor and because selective enrichment of chromatin-associated targets provides the basis of many important epigenetic bioassays. 24 After manual cell lysis and enzymatic chromatin digestion to increase the accessibility of GFP-H2B, we added mCherry to the cell lysate as an off-target fluorescent protein to be removed during washing in addition to other, non-fluorescent lysate components. Anti-GFP antibody-functionalized magnetic particles selectively captured GFP-H2B prior to magnetic washing.…”

“…13,18 To empower these capabilities in integrated droplet-based microfluidic devices, magnetic fields have provided a popular choice of flow-orthogonal force for selective manipulation of the solid phase. 4,13,16 Successful techniques include magnetic droplet translation through a series of co-flowing laminar reagent streams for applying polyelectrolyte surface coatings, 22,23 magnetic tweezers for immobilizing bead volumes while exchanging reagent droplets around them for multistep bioassays, 24,25 and additional examples of ferrofluid dispersed or continuous phase components for droplet generation and manipulation. 26,27 These approaches have demonstrated efficient and selective magnetic phase manipulation for a range of applications, but additional capabilities are needed to fully adapt solid phase (bio)chemical techniques into droplet microfluidics.…”

“…Recent reviews profile the breadth of solid phase manipulations in droplets, including alternative washing technologies to the continuously flowing droplet systems described here. 4,13 These alternatives include micropillar and microwell-based devices, 38,39 acoustic and magnetic systems for temporary bead trapping (like those described above), 24,25,40 and digital microfluidic (DMF) strategies. 41 Such techniques, however, sacrifice the throughput and mixing characteristics common to continuously flowing droplet systems.…”