A downpour of single-cell and single-molecule multiomics methods are prompting us to exclaim, “Let it rain!” We recently told you about a couple of single-molecule epigenetic mapping methods involving chromatin. And now, two new papers show how using a droplet-based microfluidic platform can provide multiomic information about single-cell transcriptomes along with histone modifications or RNA N6-methyladenosine (m6A) methylomes.
Droplet Paired-Tag: Transcriptomes and Histone Mods
Paired-Tag combines single-cell ChIP-seq and RNA-seq. It’s been around for a while, but the field has been slow on the uptake because some thought the method took too long and was too complicated. So, Bing Ren’s lab (University of California, San Diego) went back to the bench and whipped up Droplet Paired-Tag using a microfluidic platform. Forecasts indicate that the new approach will take less than 1.5 days, compared to 3 days for the old method, and can be easily adapted for use with other strategies. It also works better. Here are the details:
- Droplet Paired-Tag still involves permeabilizing nuclei and performing targeted tagmentation with antibodies that target protein A-Tn5 to histone modifications
- But here, a microfluidic device packages the nuclei and barcoded beads in droplets
- Reverse transcription (RT) and ligation happens right in the droplets with a barcoded oligonucleotide that labels cDNA, and another oligonucleotide that captures and labels tagmentation products
- All the RT and tagmentation products from each cell get the same barcode
Tests in mouse embryonic stem cells showed the method was comparable to Paired-Tag, and was even more sensitive and specific with mouse frontal cortex tissue. The new method also could identify cis-regulatory elements and their targets, as well as provide better info on their chromatin state and expression.
Single-Nucleus m6A-CUT&Tag (sn-m6A-CT): Transcriptomes and RNA Mods
Droplets are also in the forecast for analyzing m6A RNA modifications in single nuclei. Most current m6A methods don’t focus on single cells, involve harsh conditions, and are labor-intensive. But Yuin-Han Loh’s lab (A*STAR, National University of Singapore) realized that using a microfluidic device would make things go swimmingly. Their new sn-m6A-CT is a modified CUT&Tag protocol for single nuclei that can easily be scaled up. No sample prep is needed, and m6A RNA can be enriched and analyzed in situ. Here’s the info:
- First, they developed m6A-CT, and like CUT&Tag and Droplet Paired-Tag, targeted tagmentation is done with antibodies—this time, targeting m6A RNAs
- m6A antibodies bind to the RNA in isolated nuclei, then secondary antibodies bind, and then Tn5 conjugated to protein A/G binds
- RT and tagmentation are performed simultaneously to get gene expression and m6A info
- Then, to go down to the single-nucleus level, they developed sn- m6A-CT using a droplet-based microfluidic device so that m6A and unmodified RNAs from individual cells can be captured and analyzed
Tests with mouse embryonic stem cells and human cells showed that it is a sensitive and robust high-throughput approach, and the results were similar to those obtained with other methods. sn-m6A-CT allowed the team to separate out different cell types from a heterogeneous population, and they could obtain cell-type-specific m6A profiles.
Showers of Epigenetic Data in Your Future?
Although rain showers usually put a damper on plans, these new methods show that droplets in the lab are a good thing! Microfluidic devices produce a fine spray that can drill down to the single-cell and single-nucleus level to provide even more fine-tuned insights on RNA and histone modifications in the context of gene expression.
Time to put on a raincoat and galoshes and splash over to Droplet Paired-Tag (Nature Structural & Molecular Biology, August 2023) and sn-m6A-CT (Molecular Cell, September 2023).