Fig. 1: Experimental outline of ARTseq-FISH.
From: ARTseq-FISH reveals position-dependent differences in gene expression of micropatterned mESCs
a The experimental workflow of ARTseq-FISH enables the detection of different molecular species (mRNA, proteins and phosphoproteins) simultaneously. Each antibody conjugated to oligonucleotides, targets its associated (phospho-)protein in situ. Target-specific PLPs bind to these oligonucleotides as well as cellular mRNA, respectively. Then the PLPs are circularised and amplified into RCPs. A set of target-specific BrPs hybridise to RCPs, and then RoPs labelled with fluorophores bind to one of the four flanking sequences on the BrP. b Representative raw data of simultaneously detecting Beta-catenin mRNA, protein, and phosphoprotein with the same resolution. Scale bar, 20 µm, 1 µm. c The relative mean pixel intensity of the hybridisation signal’s point spread functions (n = 2000) around the local maxima. Comparison of the average observed signal of ARTseq-FISH to smFISH and a 200 nm fluorescent bead. d The sequential stripping and rehybridisation of new known RoPs allows subsequent decoding of stacked signals. Source data are provided as Source Data sheet tab Fig. 1c.
