Fig. 1: PHOTON enables spatially resolved transcriptome profiling.

A Schematic diagram illustrating the PHOTON method. First, in situ RT is performed using photocleavable RT primers. Second, targeted illumination is performed within specific ROIs identified by fluorescence imaging. Exposure of the cDNA molecules to near-UV light modifies the N-POM-caged dTs and breaks the photocleavable linkers on the RT primers, releasing the fluorescent labels and revealing the phosphate groups. Third, nucleic acids are purified, and PCR handles are ligated to previously photocleaved cDNA molecules through splint oligos. Finally, successfully ligated cDNA molecules are pulled down using streptavidin beads, and a template switching step is performed with TSOs. Following PCR amplification, sequencing libraries are generated and read by next-generation sequencing. B Design of the RT primer. C Photocleavage of cDNA molecules in HeLa cells. The color-outlined cells were exposed to 25 mW 405 nm laser light, which cleaved the fluorophores from the RT primers and resulted in an 86.9% ± 4.3% (mean ± s.d.) intensity decrease (n  =  51 cells, 1 experiment). Scale bar, 5 µm. The box represents the range between the first and third quartiles of the data. The line within the box indicates the median. The whiskers extend to the furthest data points. Source data are provided as a Source Data file. D Measuring the efficiency of the PHOTON photocaging mechanism. Left: tapestation gel image of a representative experiment; Right: bar graph showing the photocaging efficiency of PHOTON (n  =  3 independent experiments). On average, 99.1 ± 0.26% (mean ± s.d.) of the photocaged cDNA molecules could not be amplified without near-UV light exposure. Source data are provided as a Source Data file. E SNR of PHOTON as a function of the fraction of cells selected under two different laser power conditions. The shaded areas represent the 95% confidence interval. Source data are provided as a Source Data file.