Supplementary Figure 5: Evaluation of blocking buffer on non-specific binding of AbB to cells.
From: Multiplexed quantification of proteins and transcripts in single cells
(a) PBMCs were blocked with either DNA salmon sperm (1 mg/ml), dextran sulfate (0.2 mg/ml), or polyanionic Inhibitor (1 μM). Bulk PBMCs were labeled with an AbB mix (n=28) and protein libraries were prepared for bulk cells rather than single cells (less expensive for initial optimization experiments). The table shows normalized counts (AbB counts/total AbB counts x 1×104) of DNA barcodes from isotype control antibodies; Mouse IgG1, Mouse IgG2b, and Rat IgG1. Dextran sulfate showed the best reduction in non-specific binding of the AbB isotype controls. (b) PBMCs were either blocked with dextran sulfate (0.2 mg/ml) or not blocked and then labeled with an AbB mix (n=45). UMI count graphs showing the % total cells (# cells that had a specific number of UMI counts/ total # cells × 100) that are expressing a specific # of UMI counts. Single cells (without DS, n=3,158, with DS, n=4,330) were processed with REAP-seq and protein measurements show that dextran sulfate blocking helped reduce non-specific binding of the isotype controls and increased the % cells with 0 UMI counts. All three isotype controls blocked with dextran sulfate had a background noise of <= 2 UMI counts in >96% of the cells.
