Abstract
Current approaches of single cell DNA-RNA integrated sequencing are difficult to call SNPs, because a large amount of DNA and RNA is lost during DNA-RNA separation. Here, we performed simultaneous single-cell exome and transcriptome sequencing on individual mouse oocytes. Using microinjection, we kept the nuclei intact to avoid DNA loss, while retaining the cytoplasm inside the cell membrane, to maximize the amount of DNA and RNA captured from the single cell. We then conducted exome-sequencing on the isolated nuclei and mRNA-sequencing on the enucleated cytoplasm. For single oocytes, exome-seq can cover up to 92% of exome region with an average sequencing depth of 10+, while mRNA-sequencing reveals more than 10,000 expressed genes in enucleated cytoplasm, with similar performance for intact oocytes. This approach provides unprecedented opportunities to study DNA-RNA regulation, such as RNA editing at single nucleotide level in oocytes. In future, this method can also be applied to other large cells, including neurons, large dendritic cells and large tumour cells for integrated exome and transcriptome sequencing.
Highlights
Integrated single-cell exome and transcriptome data can address many questions, including somatic variation, meiotic recombination, cell-to-cell heterogeneity in gene expression and DNA-RNA regulation
RNA editing in mRNA alters the amino acid sequence of the encoded protein so that it differs from the protein predicted by the genomic DNA sequence
The editing events can be divided into two categories, one called insertion/ deletion editing, and the other called substitution of nucleotides within the RNA molecule caused by an adenosine deaminase (ADAR) enzyme[6]
Summary
These studies presented significant technical advances in simultaneous single-cell genome/transcriptome profiling They successfully separated RNA from genomic DNA (gDNA) of the same cell, either by capturing mRNA with magnetic beads and collecting gDNA from lysed supernatants[1], or by releasing the cytoplasm from the cell while keeping the nucleus completely intact[2]. They used comparative genomic hybridization and cDNA array analysis[1], or targeted sequencing of selected genes and transcripts[2], to reveal the connections between the genotype and phenotype of a single cell. By integrating the exome and transcriptome profiles in a single cell, we obtained informative results on RNA editing, which shed light on the connection between genotype and phenotype of a single cell
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