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Single-cell multi-omics are powerful means to study cell-to-cell heterogeneity. Here, we present a single-tube, bisulfite-free method for the simultaneous, genome-wide analysis of DNA methylation and genetic variants in single cells: epigenomics and genomics of single cells analyzed by restriction (epi-gSCAR). By applying this method, we obtained DNA methylation measurements of up to 506,063 CpGs and up to 1,244,188 single-nucleotide variants from single acute myeloid leukemia-derived cells. We demonstrate that epi-gSCAR generates accurate and reproducible measurements of DNA methylation and allows to differentiate between cell lines based on the DNA methylation and genetic profiles.
In conclusion, epi-gSCAR is a multistep bisulfite-free single-tube workflow for the simultaneous and genome-wide characterization of DNA methylation and genetic variants in single cells. epi-gSCAR can readily be applied to assess the phenotypic and genotypic characteristics of single cells, and has the potential to be easily integrated in current and future single-cell assays to further expand the applications of multimodal profiling of single cells, for example, in cancer.
Publicly available MALBAC and MDA datasets were downloaded from the European Nucleotide Archive (SRS2062840; MALBAC, Yikon, Single Cell YK5) and the Sequence Read Archive (SRR617646; MALBAC, sw480 single cell9, and SRR5219394; Qiagen Repli-g MDA29), mapped with BWA-MEM to the human reference genome GRCh37 (hg19). Both MALBAC datasets were downsampled to read numbers comparable to the epi-gSCAR libraries prior to mapping.
To perform ex vivo studies, human cord blood units were provided by the CORD:USE Cord Blood Bank (Orlando, FL, USA). All cord blood samples were screened for alanine aminotransferase (ALT) and pathogenic antigen antibodies (including anti-HCV, anti-HBsAg, anti-HIV, anti-Syphilis, anti-Chlamydia, and anti-Gonorrhea Abs), and only pathogen-free cord blood units were used for isolating CB-SCs. Human cord blood-derived stem cells (CB-SCs) were generated as previously described [24,25] with the following modifications. Cord blood mononuclear cells were plated in serum-free culture medium (Lonza, Walkersville, MD, USA) and incubated at 37°C, in 8% CO2. After 2 to 3 weeks, CB-SCs growing at 80-90% confluence were prepared for co-culture with allogeneic lymphocytes. 2b1af7f3a8