Development of a droplet-microfluidic system for single cell sequencing of microRNAs
Paige Coles, Salil Garg, Suman Bose, Jeremy Caplin, Vikash Pal Singh Chauhan, Andrew Bader, Anthony Chui, Robert Langer, Daniel Anderson, Phillip Sharp
Koch Institute for Integrative Cancer Research at MIT
Our goal is to utilize barcoding bead technology1 in combination with a droplet-microfluidics platform to isolate microRNAs (miRNAs) from single cells.MiRNAs regulate mRNA transcript levels through binding to the protein Argonaute 2 (Ago-2) and correlate strongly with leukemic phenotype in peripheral blood cells. Our approach utilizes droplets in which we encapsulate and lyse single cells, immunoaffinity purify miRNA-bound Ago-2 using magnetic beads, and ligate released miRNA to DNA barcodes for sequencing.
Here we present our initial work optimizing a buffer compatible with the processes of lysis, capture, and ligation in droplets. Each component of this buffer has been optimized, including a specific combination of protease inhibitors that prevents degradation of Ago-2. We also show proof of concept results indicating successful cell lysis and ligation of synthetic miRNAs in droplets.Additionally, we show microfluidic device designs optimized for maximal capture of miRNA-bound Ago-2 in droplets. Current areas of focus are further optimization of capture in droplets and ligation in droplets of miRNA derived from total cellular RNA. Our initial attempts at ligation in droplets with total cellular RNA indicate that it will be critical to separate miRNAs from other RNAs, such as abundant ribosomal rRNA, to successfully capture a significant number of events.
1. Klein, A. M., Mazutis, L., Akartuna, I., Tallapragada, N., Veres, A., Li, V., … Kirschner, M. W. (2015). Droplet Barcoding for Single-Cell Transcriptomics Applied to Embryonic Stem Cells. Cell, 161(5), 1187–1201.
This work is supported by the Koch Institute Frontier Research Program’s Mission: Possible Award, through the Stanley M. and Lois S. Proctor Cancer Research Fund, the Jean and Sidney Silber Cancer Research Fund, and the High Priority Cancer Research Fund (Anonymous).