Question: How do I minimize background RNA during sample prep?
Answer: Cell-free, or background, RNA in single cell suspensions will (in addition to the cells) be captured in single partitions and as a result can significantly increase the background in single cell data. Optimal performance of the 10x Single Cell assay is achieved if free floating cell-free RNA is kept at minimum.
When preparing single cell suspensions, we recommend these best practices to minimize cell-free RNA:
- Minimize time between start of sample preparation and chip loading to prevent cell lysing.
- Cells can be treated with eukaryotic RNases to remove cell-free RNA, followed by washing at least 2x with PBS + 0.04% BSA to remove residual RNases prior loading cells in the Master Mix
- RNAse inhibitor can be added to the wash and resuspension buffer. We have tested Protector RNase Inhibitor from Sigma-Aldrich (Part Number 3335399001) in the recommended wash and resuspension for nuclei isolation (1X PBS with 1.0% BSA and 0.2U/µl RNase Inhibitor) with no adverse affects on assay performance.
- Cell viability should be > 90% to minimize the amount of RNA from lysed cells
Products: Single Cell 3', VDJ