Usually, you don’t want your genome sequencing served up with a sugar coating; however, thanks to a clever a new 5hmC sequencing technique, we can indulge in some sweet single-cell and strand-specific insight.
The study of 5hmC (5-Hydroxymethylcytosine) prevalence in DNA typically involves utilizing modified bisulfite sequencing, 5hmC specific antibodies, or restriction enzymes. Aba-seq makes use of the sweet-toothed restriction enzyme, AbaSI, which specifically digests glucosylated 5hmC.
Now, Aba-seq has gotten a sweet upgrade by the lab of Alexander van Oudenaarden from the Hubrecht Institute in the Netherlands who bring forth single-cell Aba-seq (scAba-seq).
Here’s the scAba-seq recipe:
- Sort single-cells into 384 well plates.
- Glucosylate 5hmC using a T4 phage β-glucosyltransferase.
- Digest glucosylated 5hmC with the restriction enzyme AbaSI to leave a sticky end.
- Ligate to an adaptor containing a:
- Single-cell specific barcode
- Illumina 5’ adaptor
- T7 promoter
- Pool and amplify the ligated DNA by in vitro transcription, making use of T7 polymerase to linearly amplify the DNA in a strand-specific manner.
- Generate directional RNA-seq libraries using the amplified RNA, with single-cell specific adaptors.
The team demonstrated the power of scAba-seq by examining mouse embryos, where they observed pronounced cell-to-cell heterogeneity in the chromosome-wide distribution of 5hmC on the plus and minus strands.
Since 5hmC isn’t maintained like 5mC (by the maintenance methyltransferase DNMT1), they reasoned that the plus and minus strand-specific skew is a result of differences in chromosome strand age. This skew stems from sister cells inheriting one old strand from the mother cell and the need to synthesize a new complimentary strand. To prove their point, they used scAba-seq to reconstruct the lineages of two- and four-cell mouse embryos.
Go sink your single-cell sweet tooth into 5hmC over at Nature Biotechnology, June 2016.