Good permeabilized cells are worth the effort
Practice the cell permeabilization until your cells are:
- High % permeabilized
- Low % non-permeabilized
- have fairly uniform appearance
- are free of debris and clumps
- In single cell suspension before they are frozen
High quality cells allow accurate normalization and spiking and ensure that each cell runs on with similar efficiency.
Minimize batch variation
- Batch cells for permeabilization carefully (see our permeabilization protocol).
- Samples intended for comparison should be prepared at the same time or as close together as possible (same or consecutive days), by the same person, using the same stocks of reagents.
- To compare new to old data most accurately, consider re-preparing controls from the old data as part of the new set
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If you think you may want to add more replicates to your experiment later, consider preparing them all together
- For example: you plan to submit duplicates but may want to add a triplicate later. In this case, prepare in triplicate but only submit the duplicates.
Cell health matters
- Keep confluency consistent. Cells should be as similar as possible with respect to health and confluency at the time of harvest. Overgrowth may lead to cell stress related transcriptome changes confounding your experimental conditions. To minimize this, seed faster growing cells at lower densities than slower growing cells.
- Keep cells happy. Cells should not be dying or lifting off dishes at the time of harvest. When health is unavoidably reduced (e.g. by knockdown of essential genes), keep the cells as healthy as possible and washed away dead cells and debris prior to permeabilization.
- Plan your timepoints. Changes in nascent RNA levels are detectable at earlier timepoints than changes in steady-state RNA. If you are having difficulty getting good viability at the time of harvest, consider looking at an earlier timepoint.