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Single-Cell Tagged Reverse Transcription (STRT-Seq)

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Single Cell Methods

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1979))

Abstract

Single-cell RNA sequencing (scRNA-seq) has become an established approach to profile entire transcriptomes of individual cells from different cell types, tissues, species, and organisms. Single-cell tagged reverse transcription sequencing (STRT-seq) is one of the early single-cell methods which utilize 5′ tag counting of transcripts. STRT-seq performed on microfluidics Fluidigm C1 platform (STRT-C1) is a flexible scRNA-seq approach that allows for accurate, sensitive and importantly molecular counting of transcripts at single-cell level. Herein, I describe the STRT-C1 method and the steps involved in capturing 96 cells across C1 microfluidics chip, cDNA synthesis, and preparing single-cell libraries for Illumina short-read sequencing.

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References

  1. Natarajan KN, Teichmann SA, Kolodziejczyk AA (2017) Single cell transcriptomics of pluripotent stem cells: reprogramming and differentiation. Curr Opin Genet Dev 46:66–76. https://doi.org/10.1016/j.gde.2017.06.003

    Article  CAS  PubMed  Google Scholar 

  2. Papalexi E, Satija R (2018) Single-cell RNA sequencing to explore immune cell heterogeneity. Nat Rev Immunol 18(1):35–45. https://doi.org/10.1038/nri.2017.76

    Article  CAS  PubMed  Google Scholar 

  3. Regev A, Teichmann SA, Lander ES, Amit I, Benoist C, Birney E, Bodenmiller B, Campbell P, Carninci P, Clatworthy M, Clevers H, Deplancke B, Dunham I, Eberwine J, Eils R, Enard W, Farmer A, Fugger L, Gottgens B, Hacohen N, Haniffa M, Hemberg M, Kim S, Klenerman P, Kriegstein A, Lein E, Linnarsson S, Lundberg E, Lundeberg J, Majumder P, Marioni JC, Merad M, Mhlanga M, Nawijn M, Netea M, Nolan G, Pe'er D, Phillipakis A, Ponting CP, Quake S, Reik W, Rozenblatt-Rosen O, Sanes J, Satija R, Schumacher TN, Shalek A, Shapiro E, Sharma P, Shin JW, Stegle O, Stratton M, Stubbington MJT, Theis FJ, Uhlen M, van Oudenaarden A, Wagner A, Watt F, Weissman J, Wold B, Xavier R, Yosef N, Human Cell Atlas Meeting P (2017) The Human Cell Atlas. Elife 6. doi:https://doi.org/10.7554/eLife.27041

  4. Rozenblatt-Rosen O, Stubbington MJT, Regev A, Teichmann SA (2017) The Human Cell Atlas: from vision to reality. Nature 550(7677):451–453. https://doi.org/10.1038/550451a

    Article  CAS  PubMed  Google Scholar 

  5. Islam S, Kjallquist U, Moliner A, Zajac P, Fan JB, Lonnerberg P, Linnarsson S (2012) Highly multiplexed and strand-specific single-cell RNA 5′ end sequencing. Nat Protoc 7(5):813–828. https://doi.org/10.1038/nprot.2012.022

    Article  CAS  PubMed  Google Scholar 

  6. Pollen AA, Nowakowski TJ, Shuga J, Wang X, Leyrat AA, Lui JH, Li N, Szpankowski L, Fowler B, Chen P, Ramalingam N, Sun G, Thu M, Norris M, Lebofsky R, Toppani D, Kemp DW 2nd, Wong M, Clerkson B, Jones BN, Wu S, Knutsson L, Alvarado B, Wang J, Weaver LS, May AP, Jones RC, Unger MA, Kriegstein AR, West JA (2014) Low-coverage single-cell mRNA sequencing reveals cellular heterogeneity and activated signaling pathways in developing cerebral cortex. Nat Biotechnol 32(10):1053–1058. https://doi.org/10.1038/nbt.2967

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Hochgerner H, Lonnerberg P, Hodge R, Mikes J, Heskol A, Hubschle H, Lin P, Picelli S, La Manno G, Ratz M, Dunne J, Husain S, Lein E, Srinivasan M, Zeisel A, Linnarsson S (2017) STRT-seq-2i: dual-index 5′ single cell and nucleus RNA-seq on an addressable microwell array. Sci Rep 7(1):16327. https://doi.org/10.1038/s41598-017-16546-4

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Islam S, Kjallquist U, Moliner A, Zajac P, Fan JB, Lonnerberg P, Linnarsson S (2011) Characterization of the single-cell transcriptional landscape by highly multiplex RNA-seq. Genome Res 21(7):1160–1167. https://doi.org/10.1101/gr.110882.110

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Zeisel A, Munoz-Manchado AB, Codeluppi S, Lonnerberg P, La Manno G, Jureus A, Marques S, Munguba H, He L, Betsholtz C, Rolny C, Castelo-Branco G, Hjerling-Leffler J, Linnarsson S (2015) Brain structure. Cell types in the mouse cortex and hippocampus revealed by single-cell RNA-seq. Science 347(6226):1138–1142. https://doi.org/10.1126/science.aaa1934

    Article  CAS  PubMed  Google Scholar 

  10. Svensson V, Natarajan KN, Ly LH, Miragaia RJ, Labalette C, Macaulay IC, Cvejic A, Teichmann SA (2017) Power analysis of single-cell RNA-sequencing experiments. Nat Methods 14(4):381–387. https://doi.org/10.1038/nmeth.4220

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Correspondence to Kedar Nath Natarajan .

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1 Electronic Supplementary Materials

Supplementary Table 1

Barcodes and oligonucleotides sequences for STRT-seq (DOCX 34 kb)

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Natarajan, K.N. (2019). Single-Cell Tagged Reverse Transcription (STRT-Seq). In: Proserpio, V. (eds) Single Cell Methods. Methods in Molecular Biology, vol 1979. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9240-9_9

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  • DOI: https://doi.org/10.1007/978-1-4939-9240-9_9

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-9239-3

  • Online ISBN: 978-1-4939-9240-9

  • eBook Packages: Springer Protocols

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