Brief Communication | Published:

Profiling the transcriptome with RNA SPOTs

Nature Methods volume 14, pages 11531155 (2017) | Download Citation

Abstract

Single-molecule FISH (smFISH) has been the gold standard for quantifying individual transcript abundances. Here, we scale up multiplexed smFISH to the transcriptome level and profile 10,212 different mRNAs from mouse fibroblast and embryonic stem cells. This method, called RNA sequential probing of targets (SPOTs), provides an accurate, flexible, and low-cost alternative to sequencing for profiling transcriptomes.

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Acknowledgements

We thank P. Sims, A. Raj, B. Wold, S. Linnarson, and R. Sandberg for discussions. We thank Y. Zhang for help with automated fluidics system, N. Koulena for helping in probe generation, and E. Lubeck for help at the early stage of the project. This work was supported by grants from US National Institutes of Health (NIHRNA-1-NIHP.HD075605 and RNASUPP-1-NIHP.HD075605).

Author information

Affiliations

  1. Division of Chemistry and Chemical Engineering, Caltech, Pasadena, CA, USA.

    • Chee-Huat Linus Eng
  2. UCLA-Caltech Medical Scientist Training Program, David Geffen School of Medicine, University of California at Los Angeles, Los Angeles, CA, USA.

    • Sheel Shah
  3. Division of Engineering and Applied Science, Caltech, Pasadena, CA, USA.

    • Julian Thomassie
  4. Division of Biology and Biological Engineering, Caltech, Pasadena, CA, USA.

    • Long Cai

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Contributions

C.-H.L.E. and L.C. conceived and designed experiments. C.-H.L.E. performed all the experiments. S.S. wrote the scripts for image analysis. C.-H.L.E. performed image analysis with the guidance of S.S. J.T. wrote the script for probe generations. C.-H.L.E. and L.C. performed data analysis. All authors contributed in writing the manuscripts.

Competing interests

L.C., C.-H.L.E., and S.S have filed a patent on RNA SPOTs and its barcoding scheme.

Corresponding author

Correspondence to Long Cai.

Integrated supplementary information

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–9 and Supplementary Table 5

  2. 2.

    Life Sciences Reporting Summary

    Life Sciences Reporting Summary

  3. 3.

    Supplementary Protocol

    RNA SPOTs (Sequential Probing of Targets)

Excel files

  1. 1.

    Supplementary Table 1

    Primary probe sequences for 10,212 genes

  2. 2.

    Supplementary Table 2

    12-base code book for 10,212 genes

  3. 3.

    Supplementary Table 3

    Readout probes sequences

  4. 4.

    Supplementary Table 4

    Spots per million in NIH/3T3 and ES-E14 cells

CSV files

  1. 1.

    Supplementary Table 6

    Differential gene expression between mouse NIH/3T3 and ES-E14 cells

Zip files

  1. 1.

    Supplementary Software

    Analysis code for RNA SPOTs

About this article

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DOI

https://doi.org/10.1038/nmeth.4500