Dropception is a microfluidic platform for creating double emulsion (water-in-oil-in-water) droplets that can be sorted in high-throughput using standard flow cytometers (FACS machines). Dropception can be used to encapsulate single cells, isolate individual rare droplets of interest in wells of a multiwell plate, and recover all encapsulated nucleic acids, enabling a wide range of novel single-cell multi-omic techniques.

Brower et al., “Optimized double emulsion flow cytometry with high-throughput single-droplet isolation”, Lab Chip 2020.
Brower*, Khariton*, et al., “Double emulsion picoreactors for high-throughput single-cell encapsulation and phenotyping via FACS”, Anal. Chem. 2020.

MRBLEs (Microspheres with Ratiometric Barcode Lanthanide Encoding) rely on spectral multiplexing to track analytes throughout an experiment. We can create microspheres containing > 1,000 distinct ratios of lanthanide nanophosphors that can be uniquely identified via imaging alone, and these MRBLEs can be used to measure protein/peptide binding affinities in high-throughput using small amounts of material.

Gerver et al., “Programmable microfluidic synthesis of spectrally encoded microspheres”, Lab on a Chip 2012.
Nguyen et al., “Programmable microfluidic synthesis of over one thousand uniquely identifiable spectral codes”, Adv. Opt. Mat 2017.
Nguyen et al., “Quantitative mapping of protein-peptide affinity landscapes using spectrally encoded beads”, eLife 2019.
Harink et al., “An open-source software package for Microspheres with Ratiometric Barcode Lanthanide Encoding (MRBLEs),” PLoS ONE 2019.
Hein et al., “Protocol for peptide synthesis on spectrally encoded beads for MRBLE-pep assays,” Bio-protocols 2020.
Feng et al., “MRBLEs 2.0: High-throughput generation of chemically functionalized spectrally and magnetically-encoded hydrogel beads using a simple single-layer microfluidic device,” Microsyst. & Nanoeng. 2020.
Feng et al., “Structure-activity mapping of the peptide- and force-dependent landscape of T-cell activation,” bioRXiv 2021.

Array-based multiplexing experiments (STAMMP and HT-MEK) employ microfluidic devices containing 1,568 valved reaction chambers aligned to printed DNA arrays to allow high-throughput expression, purification, and biophysical characterization of thousands of protein variants in hours.

Aditham et al., “High-throughput affinity measurements for mutations spanning a transcription factor-DNA interface reveal affinity and specificity determinants”, Cell Systems 2020.
Markin*, Mokhtari*, et al., “Revealing enzyme functional architecture via high-throughput microfluidic enzyme kinetics”, Science 2021.

RNA-MAP: RNA-protein and RNA-RNA interactions drive fundamental biological processes and are targets for molecular engineering, yet quantitative and comprehensive understanding of the sequence determinants of affinity remains limited. We have repurposed a high-throughput sequencing instrument to quantitatively measure binding and dissociation of a fluorescently labeled protein or RNA to >10^7 RNA targets generated on a flow-cell surface by in situ transcription and intermolecular tethering of RNA to DNA. Quantitative analysis of RNA-protein and RNA-RNA interactions on a massively parallel array (RNA-MaP) provides generalizable insight into the biophysical basis and evolutionary consequences of sequence-function relationships.

Buenrostro*, Araya*, et al., "Quantitative analysis of RNA-protein interactions on a massively parallel array reveals biophysical and evolutionary landscapes", Nat. Biotech. 2014.
She*, Chakravarty*, Layton* et al., “Comprehensive and quantitative mapping of RNA–protein interactions across a transcribed eukaryotic genome”, PNAS 2017.
Boyle et al., “High-throughput biochemical profiling reveals sequence determinants of dCas9 off-target binding and unbinding”, PNAS 2017.
Denny*, Bisaria*, et al., “High-Throughput Investigation of Diverse Junction Elements in RNA Tertiary Folding”, Cell 2018.
Denny & Greenleaf, “Linking RNA Sequence, Structure, and Function on Massively Parallel High-Throughput Sequencers .” CSH Perspectives in Biology 2018.
Jarmoskaite et al., “A Quantitative and Predictive Model for RNA Binding by Human Pumilio Proteins ” Mol.Cell 2019.
Becker, Ober-Reynolds, et al., “High-Throughput Analysis Reveals Rules for Target RNA Binding and Cleavage by AGO2” Mol. Cell 2019.
Andreasson, Savinov, Block, & Greenleaf “Comprehensive sequence-to-function mapping of cofactor-dependent RNA catalysis in the glmS ribozyme”, Nat. Comm. 2020.

Prot-MAP: High-throughput DNA sequencing techniques have enabled diverse approaches for linking DNA sequence to biochemical function. In contrast, assays of protein function have substantial limitations in terms of throughput, automation, and widespread availability. We have adapted an Illumina high-throughput sequencing chip to display an immense diversity of ribosomally translated proteins and peptides and then carried out fluorescence-based functional assays directly on this flow cell, demonstrating that a single, widely available high-throughput platform can perform both sequencing-by-synthesis and protein assays.

Layton, McMahon & Greenleaf, “Large-Scale, Quantitative Protein Assays on a High-Throughput DNA Sequencing Chip,” Mol. Cell 2019.

Massively parallel filter binding:  We developed a scalable platform for assaying protein-nucleic acid interactions and cleavage in high throughput using filter binding followed by high throughput sequencing.

Boyle et al., “Quantification of Cas9 binding and cleavage across diverse guide sequences maps landscapes of target engagement,” Sci. Advances 2021.