Single Cell Analysis Program Highlights
MASSIVELY PARALLEL POLYMERASE CLONING AND GENOME SEQUENCING OF SINGLE CELLS USING NANOLITER MICROWELLS
Genome sequencing of single cells has a variety of applications, including characterizing difficult-to-culture microorganisms and identifying somatic mutations in single cells from mammalian tissues. A major hurdle in this process is the bias in amplifying the genetic material from a single cell, a procedure known as polymerase cloning. Here we describe the microwell displacement amplification system (MIDAS), a massively parallel polymerase cloning method in which single cells are randomly distributed into hundreds to thousands of nanoliter wells and their genetic material is simultaneously amplified for shotgun sequencing.
Image courtesy of Nature Publishing Group
Dr. Christopher Love at the Massachusetts Institute of Technology and colleagues have developed techniques to label heterogeneous or homogeneous cells using “barcoding” (i.e. combinatorial application of dyes, streptavidin/biotin or antibody labels), sort the barcoded cells into small wells, and then characterize the cytokines (signaling molecules) produced by the cell(s) in each of the wells. The characterization of cytokines is accomplished by a novel method, termed “microengraving,” previously developed by the authors and which uses a panel of cytokine-specific antibodies attached to a glass slide placed over the array of wells to detect cytokines. Barcoding can dramatically increase throughput of analysis and decrease reagent requirements. The authors demonstrate the utility of this approach in several different applications including constructing dose-response curves, profiling secretory responses to a variety of stimuli, and profiling secretory responses as a function of cell lineage. An intriguing, but yet unrealized, application of this method is the quantitative analysis of the communication among members in a small, well-defined group of cells contained in a single well. Such analysis may reveal fundamental insights into cell-cell interactions not obtainable through traditional methods.
Yamanaka YJ, Szeto GL, Gierahn TM, Forcier TL, Benedict KF, Brefo MS, Lauffenburger DA, Irvine DJ, Love JC. Cellular barcodes for efficiently profiling single-cell secretory responses by microengraving. Anal Chem. 2012 Dec 18;84(24):10531-6