RNA Overview
While DNA may contain the code of life, it’s RNA that allows for different cells to be and do different things. Each cell has its own unique set of “messages” that come from copying stretches of DNA templates into complementary strands of RNA. These, in turn, tell the cell which proteins to make. RNA expression is a strong indicator of which pathways are active, how much protein is being made (and which proteins are co-expressed together in the same cell), and even the cell’s sub-type. And while bulk measurements can tell a lot about a given tissue, sometimes it’s important to know what’s going on inside an individual cell rather than just an average — does every cell express 100 copies of gene X, for example, or do some make 200 copies and others make none?
With next-generation sequencing (NGS) becoming the method of choice for studying gene expression (transcriptomics), specialized platforms from companies like 10X Genomics and Bio-Rad have arisen that allow for the sequencing of thousands of individual cells in a single experiment. Robust statistics can be generated to power inferences about metabolic pathways and systems biology, differentiation and specialization, co-expression, and form-and-function relationships. Yet labs often lack the budget or infrastructure to benefit from such tools.
How RNA Works
Drop-seq, in its essence, is an RNA NGS (RNA-seq) library preparation protocol that begins by encapsulating a single cell and a uniquely barcoded bead together. Using a microfluidic device powered by three syringe pumps, individual cells, oil, and beads in lysis buffer are brought together into droplets. Inside the droplet the cells lyse and the mRNA hybridizes to oligonucleotides covalently attached to the beads. The oligos not only capture the RNA but serve as a template for the subsequent reactions that incorporate sequences necessary for NGS. The droplets are broken open and the bound RNA is reverse-transcribed into cDNA to form barcoded STAMPs (single-cell transcriptomes attached to microparticles). STAMPs are then used as templates for PCR amplification, resulting in an expression library, each sequence of which is tagged with a barcode mapping it back to the cell of origin.
RNA Set Up
Primary author Evan Macosko tells me that you don’t have to be a microfluidics expert to set up or run the open-source Drop-Seq. The PDMS devices (chips) themselves can be purchased from a variety of companies listed in the protocol. Likewise, the barcoded beads can be obtained from Chemgenes (http://www.chemgenes.com) by asking for the beads used in Macosko et al.
Since the protocol calls for several different sizes of syringes, pushing fluids of different viscosities, and altering the flow rates from the set-up to the running of the experiments, be sure your syringe pumps have the appropriate capabilities.
It should only take a couple of weeks to get things up and running and start generating usable data, Macosko says. In the process, it’s imperative to do a species-mixing experiment to validate that the system produces libraries with organism-specific barcodes – indicating that you’ve achieved high single-cell integrity.