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Photolabile oligonucleotides with topological light gradients allow spatially dealt with single-cell transcriptomics and epigenomics

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Photolabile oligonucleotides with topological light gradients allow spatially dealt with single-cell transcriptomics and epigenomics

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Data schedule 19460850 Raw and processed sequencing information produced in this research study were transferred to the National Center for Biotechnology Information Gene Expression Omnibus under accession number GSE237524 Source information are offered with this paper.

Code accessibility 19460851 Customized code for evaluating scSTAMP-seq information and the accompanying paperwork are offered on GitHub (https://github.com/deylabucsb/scSTAMP-seq. 19460855 19460855

References

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    Acknowledgements 19460850 We thank members of the S.S.D. and M.Z.W. laboratories for useful conversations. We thank J. Smith at the Biological Nanostructures Laboratory in the CNSI, supported by UCSB and UC Office of the President, for assist with Illumina sequencing, and T. Parsons at Nikon Instruments for support with microscopy. We thank the D. Acosta-Alvear laboratory( UCSB) for supplying HeLa-mCherry cells. We thank the K. S. Kosik laboratory( UCSB) for supplying access to their 10x Genomics Chromium gadget.

    Funding 19460850 19460851 Computational work was supported by the Center for Scientific Computing at CNSI and Material Research Laboratory at UCSB through the National Science Foundation( NSF; Materials Research Science and Engineering Centers program DMR-1720256 and CNS-1725797). This work was supported by California Institute for Regenerative Medicine Research Training Grant EDUC4-12821 to R.A.P., a Dow Discovery Fellowship to C.W., an Arnold and Mabel Beckman Foundation Scholars Award to M.G., United States Army Research Office agreement W911NF-19-D-0001 and cooperative arrangement W911NF-19-2-0026 for the Institute for Collaborative Biotechnologies to M.Z.W. and the UCSB CNSI Seed-Tech award, National Institutes of Health grants R01HD099517 and R01HG011013 and NSF grant 2339849 to S.S.D. 19460854

    Author info 19460850 19460851 Author notes 19461182 19460851 These authors contributed similarly: Robert A. Piscopio, Alex Chialastri, Chieh Wang, Mikolaj Godzik. 19461171 Authors and Affiliations 19461185 19460851 Department of Molecular, Cellular, and Developmental Biology, University of California, Santa Barbara, Santa Barbara, CA, USA 19460851 Robert A. Piscopio, Mikolaj Godzik, Weiyue Wang, Lauren J. Li & Maxwell Z. Wilson Department of Chemical Engineering, University of California, Santa Barbara, Santa Barbara, CA, USA 19460854 19460851 Alex Chialastri, Chieh Wang, Kellie A. Heom, Weiyue Wang, Neha Saxena & Siddharth S. Dey 19460854 19460851 Department of Bioengineering, University of California, Santa Barbara, Santa Barbara, CA, USA 19460851 Neha Saxena & Siddharth S. Dey 19460854 19460873 Neuroscience Research Institute, University of California, Santa Barbara, Santa Barbara, CA, USA 19460851 Maxwell Z. Wilson & Siddharth S. Dey 19461191 Authors 19460854 Robert A. Piscopio 19461181 19460873 Alex Chialastri 19460873 Chieh Wang 19461191 Mikolaj Godzik 19461181 19460873 Kellie A. Heom 19460873 19461191 Weiyue Wang 19460873 Lauren J. Li 19461181 19460873 Neha Saxena Maxwell Z. Wilson 19461181 19460873 Siddharth S. Dey 19461181 19460873 19461171 Contributions Concept, R.A.P., A.C., C.W., M.Z.W. and S.S.D. Methodology, R.A.P., A.C., C.W., M.Z.W. and S.S.D. Investigation, R.A.P., A.C., C.W., M.G., W.W., L.J.L. and N.S. Formal analysis, R.A.P., A.C., C.W., M.G. and K.H. Writing– initial draft, R.A.P. Writing– evaluation and modifying, R.A.P., A.C., C.W., M.G., M.Z.W. and S.S.D. Funding acquisition, S.S.D. Resources, M.Z.W. and S.S.D. Supervision, M.Z.W. and S.S.D. Corresponding authors 19461185 Correspondence to Maxwell Z. Wilson or Siddharth S. Dey. 19460854

    Ethics statements 19460850 Contending interests R.A.P., A.C., M.Z.W. and S.S.D. are creators on a patent application for this technique. The other authors state no completing interests. 19460855 19460855

    Additional info 19460850 Publisher’s note 19460869 Springer Nature stays neutral with regard to jurisdictional claims in released maps and institutional associations. 19460855

    Extended information 19460850

    19461215 Extended Data Fig. 1 Design of various PHO versions and downstream capture in single-cell sequencing. 19460851 a , Schematic of the parts of PHO and MPO particles. Both the ‘inner’ and ‘external’ DNA series of PHOs include a PCR manage for downstream amplification, an ‘inner’ – and ‘external’ – particular barcode, a 6-nucleotide UMI to count specific particles, and a poly-A capture series. The ‘inner’ PCR manage likewise hybridizes to a complementary series on the CMO anchor to embed in cell membranes. MPOs look like the ‘inner’ PHO series however include an extra multiplex recognition series to differentiate MPOs from PHOs. b 19460869, Schematic of 3 PHO fluorophore conjugation plans. PHOs can be conjugated to ‘inner’ and ‘external’ fluorophores, an ‘external’ fluorophore alone, or no fluorophore to allow visualization of PHO cleavage prior to downstream sequencing. 19460868 c 19460869, Schematic of PHO library preparation. Uncleaved and cleaved PHOs are very first copied utilizing a guide consisting of a T7 promoter (teal), RA5 manage (purple), cell-specific barcode, UMI (magenta), and polyT capture series (gray). Following 2nd hair synthesis, PHO-derived particles are separated from endogenous cDNA by SPRI bead filtration. An RA3 manage guide (yellow) together with either an ‘inner’ (blue) or ‘external’ (red) adapter and an Illumina P5 guide (indigo) are then utilized to enhance and extend PHO particles by PCR. A 2nd PCR utilizing a guide including an Illumina P7 (brown), sample index series (tan), and RA3 adapter, along with the very same RA5-P5 guide from the previous action are utilized to produce the last Illumina sequencing libraries. 19460855

    Extended Data Fig. 2 Patterned lighting of PHO identified cells. 19461185 19460868 a , Images of user-defined patterned lighting over live cells. DMD-projected photomasks (left) for a binary UCSB logo design (top) and a direct gradient (bottom) utilized to light up live cells identified with an ‘inner’ Cy3 – and ‘external’ Cy5-conjugated PHO. Corresponding ‘inner’ Cy3 (middle) and ‘external’ Cy5 (right) fluorescence images are revealed after lighting. Scale bars, 100 µm. b , Normalized Cy5 over Cy3 fluorescence as a function of DLP light strength for the direct gradient at the defined lighting times in the time lapse displayed in Supplementary Video 2. 19460868 c 19460869, d 19460869, 365-nm LED floodlight lighting over live cells. Agent Cy3 and Cy5 PHO fluorescence images (c) and matching metrology of stabilized Cy5 fluorescence (d) following 0 -, 1 -, or 10-minute floodlight lighting. Scale bars, 100 µm. In panel d , information represents 3 imaging field of visions gathered from each individually cured culture plate. Lines show the mean, boxes signify the interquartile variety (IQR), and hairs extend to the minimum and optimum worths that are within 1.5 x IQR. One culture plate was examined per lighting condition. e , DMDs can identify private cells with user-defined lighting. Agent composite fluorescence pictures of an ‘external’ Cy5-conjugated PHO-labeled (gray) co-culture consisting of wildtype U2OS and mCherry-expressing HeLa cells (orange). DMD-generated photomasks (magenta) were produced by thresholding on mCherry-positive HeLa cells. Scale bars, 100 µm. 19460868 f , Violin plot of the ratio of Cy5 fluorescence after and before lighting for HeLa and U2OS cells. Specific points represent single cells (HeLa, n=79 cells; U2OS, n=552 cells; N=631 overall cells) measured from one well of a 96-well plate. *** shows a statistically considerable distinction in between circulations ( p , The DMD-projected lighting pattern utilized for each gastruloid nest, with optimum light strength at the edge and non-linear decay towards the. f 19460869, Quantification of the lighting gradient displayed in panel ( 19460868 e. The light strength is outlined as a function of range from the edge of the lighting pattern. 19460868 g 19460869, Expected PHO cleavage from the light gradient throughout the gastruloid. A non-linear light decay profile was chosen for the lighting pattern to produce direct photocleavage of PHOs throughout radial position, consequently making the most of the vibrant variety and spatial details in the downstream readout. 19460868 h , Spatial gene expression profiles of canonical family tree markers 19460866 CDX2 , 19460866 SOX17 , EOMES , and DNMT3B throughout stabilized radial position in 2D human gastruloids. i 19460869, LOESS-smoothed expression profiles for private genes organized by resemblance in spatial expression patterns throughout the radial axis of 2D human gastruloids. Each panel represents among 4 spatial expression groups (I-IV), with z-score stabilized expression of each gene outlined as a function of stabilized range (based upon PHO cleavage ratio) from the nest edge. Expression information was assessed at 20 uniformly spaced spatial bins along the x-axis, making it possible for constant contrast throughout genes. Vibrant colored lines represent the group average, and thin lines represent expression profiles of private genes. 19460868 j 19460869, Spatial expression profiles of chromatin regulators in 2D human gastruloid nests. LOESS-smoothed spatial expression profiles of chromatin regulators. z-score stabilized expression of each gene is outlined as a function of stabilized range (based upon PHO cleavage ratio) from the nest edge. Expression information was assessed at 20 uniformly spaced spatial bins along the x-axis, making it possible for constant contrast throughout genes. The chromatin regulators are colored by classification: histone editors (red), histone readers (orange), DNA editors (blue), DNA readers (green), ATP-dependent chromatin remodelers (yellow), and Polycomb group proteins (purple). Source information 19460855

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    About this post 19460851 19460854 19460847 Mention this short article 19461185 Piscopio, R.A., Chialastri, A., Wang, C. 19460866 et al. Photolabile oligonucleotides with topological light gradients make it possible for spatially fixed single-cell transcriptomics and epigenomics. 19460866 Nat Biotechnol (2026). https://doi.org/10.1038/s41587-026-03328-5 19460854 Download citation Gotten : 19461181 19461191 16 August 2023 19461245 Accepted : 19461181 19461191 01 September 2026 19460854 19461243 19460851 Released 19461191: 19461191 25 September 2026 19461245 19460854 19460851 Variation of record : 19461191 25 September 2026 19461181 DOI 19461191: 19461181 https://doi.org/10.1038/s41587-026-03328-5 19461181 19460854 19460855 19460855