Supplementary Figure 5: Protocol optimization for RNA detection with simultaneous antibody staining.

Several surface antigens are disrupted by methanol permeabilization, which requires antibody staining to be performed before this step in the PLAYR protocol. In addition, we have found that crosslinking with the BS3 crosslinker is necessary to preserve antibody staining during the PLAYR protocol, as determined in multiple experiments with different antibodies (data not shown). However, RNA is progressively degraded during the incubations that are necessary to perform the steps mentioned above. This can be avoided by permeabilization of cells in the presence of RNAse inhibitors that inhibit endogenous and exogenous RNAses. Shown are signal intensities for the ACTB transcript in monocytes as measured by PLAYR when antibody staining is performed in different buffers containing RNAsin Ribonuclease Inhibitor (RNAsin) only or in combination with ribonucleoside vanadyl complex (RVC) and polyvinylsulfonic acid (PVS). BD Perm/Wash buffer is a commercially available saponin-based buffer and was replaced with PBS + 2% saponin that performed comparably. Signals obtained when cells are permeabilized directly without antibody staining are shown as a control. We decided to omit RVC in this step from the final protocol because the reagent interferes with the staining of some antibodies. Experiments were performed on a fluorescence flow cytometer with healthy PBMCs and monocytes were gated based on Forward and Side Scatter.