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mRNA quantification using single-molecule FISH in Drosophila embryos

Nature Protocols volume 12, pages 1326–1348 (2017)Cite this article

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Abstract

Spatial information is critical to the interrogation of developmental and tissue-level regulation of gene expression. However, this information is usually lost when global mRNA levels from tissues are measured using reverse transcriptase PCR, microarray analysis or high-throughput sequencing. By contrast, single-molecule fluorescence in situ hybridization (smFISH) preserves the spatial information of the cellular mRNA content with subcellular resolution within tissues. Here we describe an smFISH protocol that allows for the quantification of single mRNAs in Drosophila embryos, using commercially available smFISH probes (e.g., short fluorescently labeled DNA oligonucleotides) in combination with wide-field epifluorescence, confocal or instant structured illumination microscopy (iSIM, a super-resolution imaging approach) and a spot-detection algorithm. Fixed Drosophila embryos are hybridized in solution with a mixture of smFISH probes, mounted onto coverslips and imaged in 3D. Individual fluorescently labeled mRNAs are then localized within tissues and counted using spot-detection software to generate quantitative, spatially resolved gene expression data sets. With minimum guidance, a graduate student can successfully implement this protocol. The smFISH procedure described here can be completed in 4–5 d.

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Figure 2: Detection of single, smFISH-hybridized mRNAs in Drosophila embryos.
Figure 4: Determining the fluorescence intensity of a single mRNA molecule.
Figure 3: Workflow of the single mRNA molecule detection using Airlocalize.
Figure 5: smFISH enables spatial and quantitative characterization of gene expression in the fly tissue.
Figure 1: Collection and fixing of fly embryos for smFISH.

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References

  1. Femino, A.M., Fay, F.S., Fogarty, K. & Singer, R.H. Visualization of single RNA transcripts in situ. Science 280, 585–590 (1998).

    Article  CAS  PubMed  Google Scholar 

  2. Raj, A., van den Bogaard, P., Rifkin, S.A., van Oudenaarden, A. & Tyagi, S. Imaging individual mRNA molecules using multiple singly labeled probes. Nat. Methods 5, 877–879 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Eswaramoorthy, P. et al. Asymmetric division and differential gene expression during a bacterial developmental program requires DivIVA. PLoS Genet. 10, e1004526 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Little, S.C., Tikhonov, M. & Gregor, T. Precise developmental gene expression arises from globally stochastic transcriptional activity. Cell 154, 789–800 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Trcek, T. et al. Drosophila germ granules are structured and contain homotypic mRNA clusters. Nat. Commun. 6, 7962 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Little, S.C., Sinsimer, K.S., Lee, J.J., Wieschaus, E.F. & Gavis, E.R. Independent and coordinate trafficking of single Drosophila germ plasm mRNAs. Nat. Cell Biol. 17, 558–568 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Xu, H., Sepulveda, L.A., Figard, L., Sokac, A.M. & Golding, I. Combining protein and mRNA quantification to decipher transcriptional regulation. Nat. Methods 12, 739–742 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Trovisco, V. et al. icoid mRNA localises to the Drosophila oocyte anterior by random Dynein-mediated transport and anchoring. Elife http://dx.doi.org/10.7554/eLife.17537 (2016).

  9. Lionnet, T. et al. A transgenic mouse for in vivo detection of endogenous labeled mRNA. Nat. Methods 8, 165–170 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. York, A.G. et al. Instant super-resolution imaging in live cells and embryos via analog image processing. Nat. Methods 10, 1122–1126 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Curd, A. et al. Construction of an instant structured illumination microscope. Methods 88, 37–47 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Halstead, J.M. et al. Translation. An RNA biosensor for imaging the first round of translation from single cells to living animals. Science 347, 1367–1671 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Lecuyer, E., Necakov, A.S., Caceres, L. & Krause, H.M. High-resolution fluorescent in situ hybridization of Drosophila embryos and tissues. CSH Protoc. 2008 http://dx.doi.org/10.1101/pdb.prot5019 (2008).

  14. Lecuyer, E., Parthasarathy, N. & Krause, H.M. Fluorescent in situ hybridization protocols in Drosophila embryos and tissues. Methods Mol. Biol. 420, 289–302 (2008).

    Article  CAS  PubMed  Google Scholar 

  15. Wilkie, G.S. & Davis, I. Visualizing mRNA by in situ Hybridization Using 'High Resolution' and Sensitive Tyramide Signal Amplification 94–97 (Academic Press, 1994).

  16. Lecuyer, E. et al. Global analysis of mRNA localization reveals a prominent role in organizing cellular architecture and function. Cell 131, 174–187 (2007).

    Article  CAS  PubMed  Google Scholar 

  17. Buxbaum, A.R., Wu, B. & Singer, R.H. Single β-actin mRNA detection in neurons reveals a mechanism for regulating its translatability. Science 343, 419–422 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Trcek, T. et al. Single-mRNA counting using fluorescent in situ hybridization in budding yeast. Nat. Protoc. 7, 408–419 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Zenklusen, D. & Singer, R.H. Analyzing mRNA expression using single mRNA resolution fluorescent in situ hybridization. Methods Enzymol. 470, 641–659 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Sinsimer, K.S., Lee, J.J., Thiberge, S.Y. & Gavis, E.R. Germ plasm anchoring is a dynamic state that requires persistent trafficking. Cell Rep. 5, 1169–1177 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Davidson, A., Parton, R.M., Rabouille, C., Weil, T.T. & Davis, I. Localized translation of gurken/TGF-α mRNA during axis specification is controlled by access to Orb/CPEB on processing bodies. Cell Rep. 14, 2451–2462 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Gandhi, S.J., Zenklusen, D., Lionnet, T. & Singer, R.H. Transcription of functionally related constitutive genes is not coordinated. Nat. Struct. Mol. Biol. 18, 27–34 (2011).

    Article  CAS  PubMed  Google Scholar 

  23. Zenklusen, D., Larson, D.R. & Singer, R.H. Single-RNA counting reveals alternative modes of gene expression in yeast. Nat. Struct. Mol. Biol. 15, 1263–1271 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Nair, G., Walton, T., Murray, J.I. & Raj, A. Gene transcription is coordinated with, but not dependent on, cell divisions during C. elegans embryonic fate specification. Development 140, 3385–3394 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Castelnuovo, M. et al. Bimodal expression of PHO84 is modulated by early termination of antisense transcription. Nat. Struct. Mol. Biol. 20, 851–858 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Larkin, J.D. & Cook, P.R. Super-resolution measurement of distance between transcription sites using RNA FISH with intronic probes. Methods 98, 150–157 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Skinner, S.O., Sepulveda, L.A., Xu, H. & Golding, I. Measuring mRNA copy number in individual Escherichia coli cells using single-molecule fluorescent in situ hybridization. Nat. Protoc. 8, 1100–1113 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Itzkovitz, S. et al. Single-molecule transcript counting of stem-cell markers in the mouse intestine. Nat. Cell Biol. 14, 106–114 (2012).

    Article  CAS  Google Scholar 

  29. Chen, K.H., Boettiger, A.N., Moffitt, J.R., Wang, S. & Zhuang, X. RNA imaging. Spatially resolved, highly multiplexed RNA profiling in single cells. Science 348, aaa6090 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Taniguchi, Y. et al. Quantifying E. coli proteome and transcriptome with single-molecule sensitivity in single cells. Science 329, 533–538 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Battich, N., Stoeger, T. & Pelkmans, L. Image-based transcriptomics in thousands of single human cells at single-molecule resolution. Nat. Methods 10, 1127–1133 (2013).

    Article  CAS  PubMed  Google Scholar 

  32. Vargas, D.Y. et al. Single-molecule imaging of transcriptionally coupled and uncoupled splicing. Cell 147, 1054–1065 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Dong, S. et al. YRA1 autoregulation requires nuclear export and cytoplasmic Edc3p-mediated degradation of its pre-mRNA. Mol. Cell 25, 559–573 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Hoyle, N.P. & Ish-Horowicz, D. Transcript processing and export kinetics are rate-limiting steps in expressing vertebrate segmentation clock genes. Proc. Natl. Acad. Sci. USA 110, E4316–E4324 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Trcek, T., Sato, H., Singer, R.H. & Maquat, L.E. Temporal and spatial characterization of nonsense-mediated mRNA decay. Genes Dev. 27, 541–551 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Batish, M., van den Bogaard, P., Kramer, F.R. & Tyagi, S. Neuronal mRNAs travel singly into dendrites. Proc. Natl. Acad. Sci. USA 109, 4645–4650 (2012).

    Article  PubMed  PubMed Central  Google Scholar 

  37. Weil, T.T. et al. Drosophila patterning is established by differential association of mRNAs with P bodies. Nat. Cell Biol. 14, 1305–1313 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Colak, D., Ji, S.J., Porse, B.T. & Jaffrey, S.R. Regulation of axon guidance by compartmentalized nonsense-mediated mRNA decay. Cell 153, 1252–1265 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Park, H.Y., Trcek, T., Wells, A.L., Chao, J.A. & Singer, R.H. An unbiased analysis method to quantify mRNA localization reveals its correlation with cell motility. Cell Rep. 1, 179–184 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Campbell, P.D., Chao, J.A., Singer, R.H. & Marlow, F.L. Dynamic visualization of transcription and RNA subcellular localization in zebrafish. Development 142, 1368–1374 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Besse, F., Lopez de Quinto, S., Marchand, V., Trucco, A. & Ephrussi, A. Drosophila PTB promotes formation of high-order RNP particles and represses oskar translation. Genes Dev. 23, 195–207 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Bahar Halpern, K. & Itzkovitz, S. Single molecule approaches for quantifying transcription and degradation rates in intact mammalian tissues. Methods 98, 134–142 (2016).

    Article  CAS  PubMed  Google Scholar 

  43. Messier, V., Zenklusen, D. & Michnick, S.W. A nutrient-responsive pathway that determines M phase timing through control of B-cyclin mRNA stability. Cell 153, 1080–1093 (2013).

    Article  CAS  PubMed  Google Scholar 

  44. Trcek, T., Larson, D.R., Moldon, A., Query, C.C. & Singer, R.H. Single-molecule mRNA decay measurements reveal promoter- regulated mRNA stability in yeast. Cell 147, 1484–1497 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Cabili, M.N. et al. Localization and abundance analysis of human lncRNAs at single-cell and single-molecule resolution. Genome Biol. 16, 20 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. van Werven, F.J. et al. Transcription of two long noncoding RNAs mediates mating-type control of gametogenesis in budding yeast. Cell 150, 1170–1181 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Deng, W., Shi, X., Tjian, R., Lionnet, T. & Singer, R.H. CASFISH: CRISPR/Cas9-mediated in situ labeling of genomic loci in fixed cells. Proc. Natl. Acad. Sci. USA 112, 11870–11875 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Lubeck, E. & Cai, L. Single-cell systems biology by super-resolution imaging and combinatorial labeling. Nat. Methods 9, 743–748 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Levsky, J.M., Shenoy, S.M., Pezo, R.C. & Singer, R.H. Single-cell gene expression profiling. Science 297, 836–840 (2002).

    Article  CAS  PubMed  Google Scholar 

  50. Lubeck, E., Coskun, A.F., Zhiyentayev, T., Ahmad, M. & Cai, L. Single-cell in situ RNA profiling by sequential hybridization. Nat. Methods 11, 360–361 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Levesque, M.J. & Raj, A. Single-chromosome transcriptional profiling reveals chromosomal gene expression regulation. Nat. Methods 10, 246–248 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Lucy, L.B. Iterative technique for rectification of observed distributions. Astron. J. 79, 745–754 (1974).

    Article  Google Scholar 

  53. Richardson, W.H. Bayesian-based iterative method of image restoration. J. Opt. Soc. Am. 62, 55–59 (1972).

    Article  Google Scholar 

  54. Tyagi, S. & Kramer, F.R. Molecular beacons: probes that fluoresce upon hybridization. Nat. Biotechnol. 14, 303–308 (1996).

    Article  CAS  PubMed  Google Scholar 

  55. Bratu, D.P. Molecular beacons light the way: imaging native mRNAs in living cells. Discov. Med. 3, 44–47 (2003).

    PubMed  Google Scholar 

  56. Mhlanga, M.M. et al. In vivo colocalisation of oskar mRNA and trans-acting proteins revealed by quantitative imaging of the Drosophila oocyte. PLoS One 4, e6241 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Chao, J.A., Patskovsky, Y., Almo, S.C. & Singer, R.H. Structural basis for the coevolution of a viral RNA-protein complex. Nat. Struct. Mol. Biol. 15, 103–105 (2008).

    Article  CAS  PubMed  Google Scholar 

  58. Hocine, S., Raymond, P., Zenklusen, D., Chao, J.A. & Singer, R.H. Single-molecule analysis of gene expression using two-color RNA labeling in live yeast. Nat. Methods 10, 119–121 (2013).

    Article  CAS  PubMed  Google Scholar 

  59. Abbaszadeh, E.K. & Gavis, E.R. Fixed and live visualization of RNAs in Drosophila oocytes and embryos. Methods 98, 34–41 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Park, H.Y., Buxbaum, A.R. & Singer, R.H. Single mRNA tracking in live cells. Methods Enzymol. 472, 387–406 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Buxbaum, A.R., Haimovich, G. & Singer, R.H. In the right place at the right time: visualizing and understanding mRNA localization. Nat. Rev. Mol. Cell Biol. 16, 95–109 (2015).

    Article  CAS  PubMed  Google Scholar 

  62. Forrest, K.M. & Gavis, E.R. Live imaging of endogenous RNA reveals a diffusion and entrapment mechanism for nanos mRNA localization in Drosophila. Curr. Biol. 13, 1159–1168 (2003).

    Article  CAS  PubMed  Google Scholar 

  63. Tyagi, S. Imaging intracellular RNA distribution and dynamics in living cells. Nat. Methods 6, 331–338 (2009).

    Article  CAS  PubMed  Google Scholar 

  64. Bertrand, E. et al. Localization of ASH1 mRNA particles in living yeast. Mol. Cell 2, 437–445 (1998).

    Article  CAS  PubMed  Google Scholar 

  65. Daigle, N. & Ellenberg, J. ambda(N)-GFP: an RNA reporter system for live-cell imaging. Nat. Methods 4, 633–636 (2007).

    Article  CAS  PubMed  Google Scholar 

  66. Garcia, J.F. & Parker, R. MS2 coat proteins bound to yeast mRNAs block 5′ to 3′ degradation and trap mRNA decay products: implications for the localization of mRNAs by MS2-MCP system. RNA 21, 1393–1395 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  67. Gustafsson, M.G. Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy. J. Microsc. 198, 82–87 (2000).

    Article  CAS  PubMed  Google Scholar 

  68. Frise, E., Hammonds, A.S. & Celniker, S.E. Systematic image-driven analysis of the spatial Drosophila embryonic expression landscape. Mol. Syst. Biol. 6, 345 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  69. Gavis, E.R. & Lehmann, R. Localization of nanos RNA controls embryonic polarity. Cell 71, 301–313 (1992).

    Article  CAS  PubMed  Google Scholar 

  70. Lehmann, R. Germ plasm biogenesis--an oskar-centric perspective. Curr. Top. Dev. Biol. 116, 679–707 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  71. Jambor, H., Brunel, C. & Ephrussi, A. Dimerization of oskar 3′ UTRs promotes hitchhiking for RNA localization in the Drosophila oocyte. RNA 17, 2049–2057 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  72. modEncode Consortium. Identification of functional elements and regulatory circuits by Drosophila modENCODE. Science 330, 1787–1797 (2010).

  73. Ashburner, M. Drosophila (Cold Spring Harbor Laboratory, 1989).

  74. Markow, T.A., Beall, S. & Matzkin, L.M. Egg size, embryonic development time and ovoviviparity in Drosophila species. J. Evol. Biol. 22, 430–434 (2009).

    Article  CAS  PubMed  Google Scholar 

  75. Gao, M. & Arkov, A.L. Next generation organelles: structure and role of germ granules in the germline. Mol. Reprod. Dev. 80, 610–623 (2013).

    Article  CAS  PubMed  Google Scholar 

  76. Mahowald, A.P. Fine structure of pole cells and polar granules in Drosophila melanogaster. J. Exp. Zool. 151, 201–215 (1962).

    Article  Google Scholar 

  77. Voronina, E., Seydoux, G., Sassone-Corsi, P. & Nagamori, I. RNA granules in germ cells. Cold Spring Harb. Perspect. Biol. 3 http://dx.doi.org/10.1101/cshperspect.a002774 (2011).

  78. Mahowald, A.P. Ultrastructural observations on oogenesis in Drosophila. J. Morphol. 137, 29–48 (1972).

    Article  CAS  PubMed  Google Scholar 

  79. Cho, W.K. et al. RNA polymerase II cluster dynamics predict mRNA output in living cells. Elife 5 http://dx.doi.org/10.7554/eLife.13617 (2016).

  80. Raj, A., Peskin, C.S., Tranchina, D., Vargas, D.Y. & Tyagi, S. Stochastic mRNA synthesis in mammalian cells. PLoS Biol. 4, e309 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  81. Lagha, M. et al. Paused Pol II coordinates tissue morphogenesis in the Drosophila embryo. Cell 153, 976–987 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  82. Levsky, J.M., Shenoy, S.M., Pezo, R.C. & Singer, R.H. Single-cell gene expression profiling. Science 297, 836–840 (2002).

    Article  CAS  PubMed  Google Scholar 

  83. Pezo, R.C. et al. Single-cell transcription site activation predicts chemotherapy response in human colorectal tumors. Cancer Res. 68, 4977–4982 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  84. Bothma, J.P., Magliocco, J. & Levine, M. The snail repressor inhibits release, not elongation, of paused Pol II in the Drosophila embryo. Curr. Biol. 21, 1571–1577 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  85. de Turris, V., Nicholson, P., Orozco, R.Z., Singer, R.H. & Muhlemann, O. Cotranscriptional effect of a premature termination codon revealed by live-cell imaging. RNA 17, 2094–2107 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank the NYU Langone Medical Center (NYULMC) Microscopy Laboratory for providing the API DeltaVision personal DV microscope, particularly Y. Deng and M. Cammer for help with the wide-field epifluorescence microscope. We thank V. Schoonderwoert (Scientific Volume Imaging) for his help with the Huygens deconvolution software. This work was supported by the Intramural Research Programs of the US National Institute of Biomedical Imaging and Bioengineering. T.T. is a Howard Hughes Medical Institute (HHMI) fellow of the Jane Coffin Childs Memorial Fund. R.L. is an HHMI investigator. Funding for T.L. was provided by the Howard Hughes Medical Institute.

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Author notes

  1. Timothée Lionnet

    Present address: Present address: Institute for Systems Genetics, Department of Cell Biology, NYU School of Medicine, New York, USA.,

  2. Tatjana Trcek, Timothée Lionnet and Hari Shroff: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Cell Biology, Howard Hughes Medical Institute, Skirball Institute of Biomolecular Medicine, NYU School of Medicine, New York, New York, USA

    Tatjana Trcek & Ruth Lehmann

  2. Transcription Imaging Consortium, Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia, USA

    Timothée Lionnet

  3. Section on High Resolution Optical Imaging, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, Maryland, USA

    Hari Shroff

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  1. Tatjana Trcek
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Contributions

T.T. and R.L. implemented quantitative smFISH in Drosophila tissue. H.S. developed iSIM. T.L. developed Airlocalize. All the authors wrote and approved the final version of the manuscript.

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Correspondence to Tatjana Trcek.

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Supplementary information

Supplementary Table 1

Supplementary Table 1. List of sequences of all smFISH probes used in this study. (☼) denotes the position of the fluorophore in the sequence. (PDF 67 kb)

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Trcek, T., Lionnet, T., Shroff, H. et al. mRNA quantification using single-molecule FISH in Drosophila embryos. Nat Protoc 12, 1326–1348 (2017). https://doi.org/10.1038/nprot.2017.030

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