Single-cell techniques (sequencing, RNA analysis, proteomics) are very powerful ways to cover yourself and your project in more data than you have ever had to deal with before. Well, that’s not the only reason you run such assays, of course: getting down to cell-by-cell variability can illuminate some key biology that’s lost when you average everything out over a larger sample. To pick one obvious case, tumor tissue is notoriously variable, since many solid tumors are as far from homogeneous tissue as you can get on that scale: collections of genomically unstable mutant lines all fighting with each other for nutrients and space.
But how do you do single-cell sequencing, anyway? The answer generally has to involve separating and physically isolating the cells so you can pick them off one by one, and that generally has involved some sort of microfluidics setup. Sending individual cells down microchannels and into their own spots to be analyzed is a problem that’s been solved in many different ways, but one fundamental problem hasn’t been: overall efficiency. One of the highest-throughput techniques is droplet microfluidics, tiny water droplets carried along in an immiscible oil matrix. That performance comes at a price, though: the machinery is complex, not exactly low-maintenance, and quite expensive. And with DNA sequencing getting more capable all the time, getting the single-cell part working is now the bottleneck, both in throughput and in cost.
With sequencing in mind, many of these techniques use some sort of DNA barcoding, the same sort of thing you’d do for a DNA-encoded library in chemical screening. You can put together a unique oligonucleotide sequence for each cell, doing it directly on the cell or on beads that get paired with the cells, and each of those has its up- and down-sides. Barcoding on the cells takes an awful lot of pooling and splitting, and the sheer number of pipetting operations to accomplish this can get overwhelming. The bead method has really only been accomplished with straight-up microfluidics.
There’s a report of a new system that might be able to sidestep that limitation, “particle-templated instant partition sequencing (PIP-seq). It uses the same sort of water-droplet-in-oil separation method that runs through all those microfluidics channels, but it’s done all at once in an Eppendorf tube. The barcoded beads and the cells (in buffer) are combined with an oil layer and vortexed on a benchtop mixer, and the resulting emulsion (if you do it right) ends up with droplets that each have a single bead and a single cell in them. If you add some heat-activated lysis reagents at the beginning, then you can lyse the cells once they’re emulsified and get sequencing – you have your cellular DNA and your barcoded DNA all paired up and ready to go. It’s a weirdly straightforward technique, and there are probably quite a few people wondering why they didn’t try it themselves.
The hope is that this technique will put the sequencing costs back at the top of the list again, which would be good news, because that’s continuing to drop (while the cost of microfluidically separating all those cells isn’t falling nearly so dramatically). The authors say that they’re hoping to democratize single-cell techniques, allowing many more such experiments to be run where previously people had to back off because of lack of funds or lack of time on the necessary equipment.
Overall, that has to be a good thing. Getting more peoples’ hands on advanced techniques is the way to get more unusual things to happen – I was just quoting Freeman Dyson in a seminar the other day to the effect that more innovations in science have come from the availability of new tools than from the advent of new ideas. Let’s see if that happens here!
