SIDR – simultaneous isolation and parallel sequencing of genomic DNA and total RNA from single cells

Simultaneous sequencing of the genome and transcriptome at the single-cell level is a powerful tool for characterizing genomic and transcriptomic variation and revealing correlative relationships. However, it remains technically challenging to analyze both the genome and transcriptome in the same cell.

Here, researchers from the Samsung Genome Institute report a novel method for simultaneous isolation of genomic DNA and total RNA (SIDR) from single cells, achieving high recovery rates with minimal cross-contamination, as is crucial for accurate description and integration of the single-cell genome and transcriptome. For reliable and efficient separation of genomic DNA and total RNA from single cells, the method uses hypotonic lysis to preserve nuclear lamina integrity and subsequently captures the cell lysate using antibody-conjugated magnetic microbeads. Evaluating the performance of this method using real-time PCR demonstrated that it efficiently recovered genomic DNA and total RNA. Thorough data quality assessments showed that DNA and RNA simultaneously fractionated by the SIDR method were suitable for genome and transcriptome sequencing analysis at the single-cell level. The integration of single-cell genome and transcriptome sequencing by SIDR (SIDR-seq) showed that genetic alterations such as copy-number and single nucleotide variations were much accurately captured by single-cell SIDR-seq compared to that by conventional single-cell RNA-seq, although copy-number variations positively correlated with the corresponding gene expression levels. These results suggest that SIDR-seq is potentially a powerful tool to reveal genetic heterogeneity and phenotypic information inferred from gene expression patterns at the single-cell level.

Principles of the SIDR method

(A) Schematic of the SIDR method. WGA, whole genome amplification. WTA, whole transcriptome amplification. (B and C) Immunostaining of the nucleus after cell lysis. Fluorescence images of MCF7 cells in isotonic (B) and hypotonic (C) conditions. The nuclear lamina, plasma membrane, and nucleus were stained by the Alexa 488-labeled anti-Lamin B2 antibody (green), CellMask (red) and DAPI (blue), respectively. (D) The effect of cell lysis on the recovery rate of cells. The recovery rate dramatically depended on whether anti-EpCAM antibody conjugated microbeads were bound to cells before or after cell lysis as indicated at the bottom of the graph. Approximately 100 MCF7 cells underwent bead binding and/or cell lysis and were magnetically recovered, except for control cells that were not bound to microbeads. The plot shows the number of cells recovered (n = 3). (E) The effect of bead binding on the solubilization of the EpCAM protein. The levels of EpCAM, beta actin, and Lamin B2 proteins in cell lysates not solubilized during cell lysis were measured by western blot (n = 3).