Collaborations work best when team members’ abilities complement each other. That’s what makes SIMPLE-seq work so well—it’s a simple, easy combination of two methods that complement each other to provide both 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) datasets in one experiment in single cells and molecules.
Continuing on the recent theme of methods that report both 5mC and 5hmC, such as DARESOME and Joint-snhmC, Chengqi Yi’s lab (Peking University) and Chenxu Zhu’s lab (New York Genome Center, Weill Cornell Medicine) teamed up to obtain data on both modifications simultaneously without bisulfite conversion.
They combined the Yi lab’s hmC-CATCH, which detects 5hmCs, with TAPS, which can detect both modifications, but requires two separate experiments to figure out which is which. Because the TAPS protocol involves blocking 5hmCs, just performing TAPS and hmc-CATCH at the same time wouldn’t work, so the team took a sequential approach, called SIMPLE-seq (“simultaneous profiling of epigenetic cytosine modifications by sequencing”). Tagmentation before cell lysis helps distinguish different cell populations with barcodes. Here’s what they do next:
- hmc-CATCH is first up—conduct ruthenate (VI) oxidation of 5hmC to 5fC; label 5fC with indanedione
- Perform primer extension to mark the 5hmC change on the complementary strand
- TAPS is next—perform TET-mediated oxidation of 5mC to 5caC; reduce 5caC via borane reduction to DHU
Both the labeled 5fC and the DHU show up as a “C-to-T” transition after PCR. Analyzing all of the data from a single experiment provides both 5hmC and 5mC information.
SIMPLE-seq revealed differences in modifications on DNA in mouse embryonic stem cells (mESCs) cultured in 2i media that keeps the cells pluripotent, versus cells grown in serum that were likely to differentiate. Serum-cultured mESCs had more 5mC, which was negatively correlated with expression when it was at transcriptional start sites. Here are some other interesting dynamic findings:
- Transcription factor binding motifs and transcription factor expression change between 2i to serum conditions on 5hmC and 5mC sites
- 5hmC is better than 5mC at separating 2i cells from those grown in serum
- TET-mediated demethylation could target some 5hmC sites for certain roles; for example, 5hmC sites near 5mC sites are more likely to have H3K27ac modification and be associated with active regulatory elements
Next, the collaborators used SIMPLE-seq to examine differentiated cells with more static modifications—human peripheral blood mononuclear cells (PBMCs)—and here’s what they learned:
- SIMPLE-seq distinguished five major clusters of cell types
- 5mC is better than 5hmC at separating PBMCs
- An analysis of 5mC and 5hmC data at single-base resolution hints at the cells’ roles
Finally, the team worked together to tackle the mouse brain cortex, which is comprised of cells that have a high level of 5hmC, and these are the main points:
- SIMPLE-seq distinguished 11 main cell types
- Both 5mC and 5hmC data are needed to separate these cell types
Because SIMPLE-seq uncovers complex modification dynamics, the team says that the method could someday help researchers better understand transient events associated with disease. They also say that, with a few tweaks, SIMPLE-seq could collaborate well with methods that detect other modifications, such as those on RNA.
Overall, SIMPLE-seq holds it own as a single-cell strategy for pinpointing 5mC and 5hmC modifications on DNA at the same time without harming the DNA or requiring excessive manipulations.
Check out this dynamic collaboration at Nature Biotechnology, February 2024.