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Single-Cell Capture, RNA-seq, and Transcriptome Analysis from the Neural Retina

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Retinal Development

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2092))

Abstract

Single-cell RNA sequencing (scRNA-seq) is an emerging technology that can address the challenge of cellular heterogeneity. In the last decade, the cost per cell has been dramatically reduced, and the throughput has been increased by 104-fold. Like many other tissues, the retina is highly heterogeneous with an estimated of over 100 subtypes of neuronal cells. Here, we describe the current techniques to perform scRNA-seq on the adult retinal tissue including retinal dissection, retinal dissociation, assessment of cell population, cDNA synthesis, library construction, and next-generation sequencing. In addition, we introduce a workflow of scRNA-seq data analysis using open-source tools.

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References

  1. Huang S (2009) Non-genetic heterogeneity of cells in development: more than just noise. Development 136(23):3853–3862. https://doi.org/10.1242/dev.035139

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Shalek AK, Satija R, Shuga J, Trombetta JJ, Gennert D, Lu D, Chen P, Gertner RS, Gaublomme JT, Yosef N, Schwartz S, Fowler B, Weaver S, Wang J, Wang X, Ding R, Raychowdhury R, Friedman N, Hacohen N, Park H, May AP, Regev A (2014) Single-cell RNA-seq reveals dynamic paracrine control of cellular variation. Nature 510(7505):363–369. https://doi.org/10.1038/nature13437

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Grun D, Lyubimova A, Kester L, Wiebrands K, Basak O, Sasaki N, Clevers H, van Oudenaarden A (2015) Single-cell messenger RNA sequencing reveals rare intestinal cell types. Nature 525(7568):251–255. https://doi.org/10.1038/nature14966

    Article  CAS  PubMed  Google Scholar 

  4. Rizvi AH, Camara PG, Kandror EK, Roberts TJ, Schieren I, Maniatis T, Rabadan R (2017) Single-cell topological RNA-seq analysis reveals insights into cellular differentiation and development. Nat Biotechnol 35(6):551–560. https://doi.org/10.1038/nbt.3854

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Treutlein B, Lee QY, Camp JG, Mall M, Koh W, Shariati SA, Sim S, Neff NF, Skotheim JM, Wernig M, Quake SR (2016) Dissecting direct reprogramming from fibroblast to neuron using single-cell RNA-seq. Nature 534(7607):391–395. https://doi.org/10.1038/nature18323

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Jaitin DA, Weiner A, Yofe I, Lara-Astiaso D, Keren-Shaul H, David E, Salame TM, Tanay A, van Oudenaarden A, Amit I (2016) Dissecting immune circuits by linking CRISPR-pooled screens with single-cell RNA-Seq. Cell 167(7):1883–1896. e1815. https://doi.org/10.1016/j.cell.2016.11.039

    Article  CAS  PubMed  Google Scholar 

  7. Macosko EZ, Basu A, Satija R, Nemesh J, Shekhar K, Goldman M, Tirosh I, Bialas AR, Kamitaki N, Martersteck EM, Trombetta JJ, Weitz DA, Sanes JR, Shalek AK, Regev A, McCarroll SA (2015) Highly parallel genome-wide expression profiling of individual cells using Nanoliter droplets. Cell 161(5):1202–1214. https://doi.org/10.1016/j.cell.2015.05.002

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Masland RH (2012) The neuronal organization of the retina. Neuron 76(2):266–280. https://doi.org/10.1016/j.neuron.2012.10.002

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Rheaume BA, Jereen A, Bolisetty M, Sajid MS, Yang Y, Renna K, Sun L, Robson P, Trakhtenberg EF (2018) Single cell transcriptome profiling of retinal ganglion cells identifies cellular subtypes. Nat Commun 9(1):2759. https://doi.org/10.1038/s41467-018-05134-3

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Shekhar K, Lapan SW, Whitney IE, Tran NM, Macosko EZ, Kowalczyk M, Adiconis X, Levin JZ, Nemesh J, Goldman M, McCarroll SA, Cepko CL, Regev A, Sanes JR (2016) Comprehensive classification of retinal bipolar neurons by single-cell transcriptomics. Cell 166(5):1308–1323. e1330. https://doi.org/10.1016/j.cell.2016.07.054

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Tang F, Barbacioru C, Wang Y, Nordman E, Lee C, Xu N, Wang X, Bodeau J, Tuch BB, Siddiqui A, Lao K, Surani MA (2009) mRNA-Seq whole-transcriptome analysis of a single cell. Nat Methods 6(5):377–382. https://doi.org/10.1038/nmeth.1315

    Article  CAS  PubMed  Google Scholar 

  12. Hedlund E, Deng Q (2018) Single-cell RNA sequencing: technical advancements and biological applications. Mol Asp Med 59:36–46. https://doi.org/10.1016/j.mam.2017.07.003

    Article  CAS  Google Scholar 

  13. Gao R, Kim C, Sei E, Foukakis T, Crosetto N, Chan LK, Srinivasan M, Zhang H, Meric-Bernstam F, Navin N (2017) Nanogrid single-nucleus RNA sequencing reveals phenotypic diversity in breast cancer. Nat Commun 8(1):228. https://doi.org/10.1038/s41467-017-00244-w

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Goldstein LD, Chen YJ, Dunne J, Mir A, Hubschle H, Guillory J, Yuan W, Zhang J, Stinson J, Jaiswal B, Pahuja KB, Mann I, Schaal T, Chan L, Anandakrishnan S, Lin CW, Espinoza P, Husain S, Shapiro H, Swaminathan K, Wei S, Srinivasan M, Seshagiri S, Modrusan Z (2017) Massively parallel nanowell-based single-cell gene expression profiling. BMC Genomics 18(1):519. https://doi.org/10.1186/s12864-017-3893-1

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Zheng GX, Terry JM, Belgrader P, Ryvkin P, Bent ZW, Wilson R, Ziraldo SB, Wheeler TD, McDermott GP, Zhu J, Gregory MT, Shuga J, Montesclaros L, Underwood JG, Masquelier DA, Nishimura SY, Schnall-Levin M, Wyatt PW, Hindson CM, Bharadwaj R, Wong A, Ness KD, Beppu LW, Deeg HJ, McFarland C, Loeb KR, Valente WJ, Ericson NG, Stevens EA, Radich JP, Mikkelsen TS, Hindson BJ, Bielas JH (2017) Massively parallel digital transcriptional profiling of single cells. Nat Commun 8:14049. https://doi.org/10.1038/ncomms14049

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Zappia L, Phipson B, Oshlack A (2018) Exploring the single-cell RNA-seq analysis landscape with the scRNA-tools database. PLoS Comput Biol 14(6):e1006245. https://doi.org/10.1371/journal.pcbi.1006245

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Stegle O, Teichmann SA, Marioni JC (2015) Computational and analytical challenges in single-cell transcriptomics. Nat Rev Genet 16(3):133–145. https://doi.org/10.1038/nrg3833

    Article  CAS  PubMed  Google Scholar 

  18. Dobin A, Davis CA, Schlesinger F, Drenkow J, Zaleski C, Jha S, Batut P, Chaisson M, Gingeras TR (2013) STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29(1):15–21. https://doi.org/10.1093/bioinformatics/bts635

    Article  CAS  PubMed  Google Scholar 

  19. Kim D, Langmead B, Salzberg SL (2015) HISAT: a fast spliced aligner with low memory requirements. Nat Methods 12(4):357–360. https://doi.org/10.1038/nmeth.3317

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Langmead B, Trapnell C, Pop M, Salzberg SL (2009) Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol 10(3):R25. https://doi.org/10.1186/gb-2009-10-3-r25

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Ilicic T, Kim JK, Kolodziejczyk AA, Bagger FO, McCarthy DJ, Marioni JC, Teichmann SA (2016) Classification of low quality cells from single-cell RNA-seq data. Genome Biol 17:29. https://doi.org/10.1186/s13059-016-0888-1

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Correspondence to Rui Chen .

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Dharmat, R., Kim, S., Li, Y., Chen, R. (2020). Single-Cell Capture, RNA-seq, and Transcriptome Analysis from the Neural Retina. In: Mao, CA. (eds) Retinal Development. Methods in Molecular Biology, vol 2092. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0175-4_12

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  • DOI: https://doi.org/10.1007/978-1-0716-0175-4_12

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0174-7

  • Online ISBN: 978-1-0716-0175-4

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