Multiomics quick start: ATAC Seq and RNA Seq in single cells

Learn how ATAC-Seq and RNA-Seq complement each other to measure both chromatin accessibility and gene expression. ATAC-Seq identifies open and closed DNA regions, while RNA-Seq directly measures which genes are actually expressed, providing a complete picture of gene regulation in single cells.

Break up tissue and gently lyse cells to keep nuclei intact. Separate nuclei from cellular debris using column filtration or flow sorting, then assess quality and quantity using an automated hemocytometer to ensure intact, dissociated nuclei. Store purified nuclei at -70°C if needed.

Pipette up to 15,000 nuclei into a 10x Chromium chip to create an oil-in-water emulsion. Single nuclei are isolated into droplets, each containing a barcoded bead that captures and uniquely labels the nucleic acids, allowing later identification of which data came from which cell.

Recover barcoded nucleotides from the Chromium chip and conduct two separate library preparation protocols to create Illumina-compatible libraries: one for ATAC-Seq and one for gene expression analysis. This produces two indexed libraries ready for quality control.

Perform qPCR to quantify library concentration and run samples on a bioanalyzer or similar instrument to verify library length matches expected sizes. These checks ensure both ATAC and RNA libraries are suitable for sequencing.

Load ATAC and RNA libraries onto separate flow cells due to their different sequencing requirements. Calculate the number of cells that can be sequenced by dividing total possible reads by reads required per cell (20,000 for gene expression and 25,000 for ATAC-Seq).