Development of Enhanced Reduced Representation Bisulfite Sequencing Method for Single-cell Methylome Analysis

Abstract

SUMMARYSingle-cell analysis provides molecular signatures to define cell identity. To characterize cell types, DNA methylation patterns are often used as a flag of internal molecular status. There are a few reports of single-cell methylome techniques that involves bisulfite conversion. However, the step often causes DNA fragmentation, which leads to severe PCR substrate reduction. Here we developed a new version of single-cell reduced representation bisulfite sequencing (scRRBS) method to recover more CpG sites to be analysed. Our method succeeded to increase of 4.1 times in sample yield and 1.6 times in CpG site coverage. Importantly, our results indicate that the obtained single-cell DNA methylation sites are homogeneous among 12 cells, thus it may provide molecular barcodes for cell types. In summary, we succeed to develop enhanced scRRBS method and it will be more useful tool to define cell identity in the near future.

Single-Cell (Multi)omics Technologies

“…After end repair and dA tailing, premethylated indexed sequencing adaptors are ligated to the Msp1-digested DNA fragments prior to bisulfite conversion; PCR is then used to amplify the product before sequencing. The use of biotinylated 5 and 3 adaptors and rescue steps with this protocol has recently been proposed to improve sample yield (153).…”

Epigenetics and tissue immunity—Translating environmental cues into functional adaptations*

“…Single-cell reduced representation bisulfite sequencing (scRRBS) has been used to decipher heterogeneity among ESC. [94][95][96] More recently, methods to simultaneously assay single-cell methylome and transcriptome have been developed, relying on physical separation of RNA and genomic DNA. These include Switching Mechanism At the end of the 5'-end of the RNA Transcript (Smart)-RRBS and scMT-seq which combine the Smart-seq2 whole transcriptome method 97 and RRBS, 98,99 scM&T-seq which adapts the existing G&T-seq approach 100 with a bisulfite conversion step for genomic DNA.…”