Extended Data Fig. 1: Alternative O-MAP designs.
a, summary of RNA-targeted HRP-recruitment strategies tested. Design 1 uses biotinylated primary probes to recruit a streptavidin-HRP conjugate. Designs 2 and 3 use Digoxigenin (DIG)-labeled primary or secondary probes to recruit an HRP–conjugated anti-DIG antibody. Our final O-MAP design, which uses HRP-conjugated oligo probes, is shown for comparison (see also, Fig. 1a). The same anti-DIG antibody is used in designs 2 and 3; the same “universal landing pad” sequences are used in designs 3 and 4. b–e, limitations of Designs 1–3. In all cases biotin was visualized by staining with a fluorescent neutravidin conjugate. b, Design 1 was disfavored because in situ biotinylation cannot be distinguished from biotinylated probes. HeLa cells over-expressing NPM1~eGFP were probed using the same 47S-targeting probes as in (Supplementary Fig. 1), bearing 3´–terminal biotin. c, Design 2—analogous to HyPro—in HeLa cells. This sometimes produced well-resolved nucleolar-targeted biotinylation, but was disfavored due to antibody irreproducibility (see below). The high cost of DIG-labeled oligos would also limit its use with low-abundance transcripts, which can require dozens-to-hundreds of probes. d, Design 3 overcomes the oligo cost issue but still suffers from antibody background binding and irreproducibility. HeLa cells were probed with the same 47S-targeting primary probe set and DIG-labeled secondary oligo, using four different lots of commercial HRP-conjugated antibodies. In some cases, we observed well-resolved RNA-targeted biotinylation (panel i), though other lots from the same vendor exhibited significant off-target labeling (panel ii, arrows). Regents from other vendors exhibited varying degrees of blurring (panel iii), or excessive off-target biotinylation (panel iv). e, Design 3 is particularly problematic with lower-abundance RNA targets. Patski cells were probed with the same Xist-targeting, landing-pad-extended probes as used in the main text, divided into two sub-pools. Anti-DIG-HRP was from Vendor 1. Note that all conditions—both probe sub-pools and the omit-primary negative control–induced substantial off-target biotinylation. All scale bars, 20 µm.
