The simultaneous sequencing of a single cell’s genome and transcriptome is powerful tools to understand the genomic and transcriptomic variations, and their correlative relationships. However, technical obstacles have prohibited the simultaneous analysis of both genome and transcriptome derived from a single cell. Here, we report a method for simultaneous isolation of DNA and total RNA (SIDR) from single cells, especially for parallel genome and transcriptome sequencing. The method adopts a strategy to physically separate genomic DNA and total RNA from single cells, based on hypotonic lysis and subsequent separation of cell lysate associated with magnetic microbeads. Systematic performance evaluation by quantitative real-time PCR and single-cell sequencing demonstrated that the method efficiency recovered genomic DNA and total RNA. We also validated that genomic DNA and total RNA simultaneously fractionated by SIDR method were suitable for the genome and transcriptome sequencing analysis of single cells, based on various aspects of sequencing data quality. By applying SIDR to integrated genome and transcriptome sequencing, this platform may be potentially an enabling tool to merge biological genetic and phenotypic information at the single cell scale.