Abstract
Chromium 10× 3′ V2 protocol is a 3′ end counting single-cell mRNA sequencing protocol that allows to process and sequence RNA from thousands of cells in parallel. Chromium10× by 10× Genomics is an emulsion-based device that enables to compartmentalize single cells along with sets of uniquely barcoded primers and reverse transcription reagents into nanoscale droplets that are used as reaction chambers to generate barcoded full-length cDNA from single cells. After RT reaction single-stranded barcoded cDNAs are pooled together and processed to generate sequencing libraries compatible with the standard Illumina platforms. Here we show in detail the main steps of the protocol applied to the analysis of tumor-infiltrating T lymphocytes (TILs). The main steps are cell preparation, cDNA synthesis, library construction, and sequencing.
This protocol refers specifically to the CG00052_SingleCell3_ReagentKitv2UserGuide_RevD downloadable from 10× Genomics website (https://www.10xgenomics.com) and does not substitute it. Always refer to this guide, paying attention to updates and revisions.
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References
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
Zhu YY, Machleder EM, Chenchik A, Li R, Siebert PD (2001) Reverse transcriptase template switching: a SMART approach for full-length cDNA library construction. BioTechniques 30(4):892–897
De Simone M, Arrigoni A, Rossetti G, Gruarin P, Ranzani V, Politano C, Bonnal RJP, Provasi E, Sarnicola ML, Panzeri I, Moro M, Crosti M, Mazzara S, Vaira V, Bosari S, Palleschi A, Santambrogio L, Bovo G, Zucchini N, Totis M, Gianotti L, Cesana G, Perego RA, Maroni N, Pisani Ceretti A, Opocher E, De Francesco R, Geginat J, Stunnenberg HG, Abrignani S, Pagani M (2016) Transcriptional landscape of human tissue lymphocytes unveils uniqueness of tumor-infiltrating T regulatory cells. Immunity 45(5):1135–1147. https://doi.org/10.1016/j.immuni.2016.10.021
Tirosh I, Izar B, Prakadan SM, Wadsworth MH 2nd, Treacy D, Trombetta JJ, Rotem A, Rodman C, Lian C, Murphy G, Fallahi-Sichani M, Dutton-Regester K, Lin JR, Cohen O, Shah P, Lu D, Genshaft AS, Hughes TK, Ziegler CG, Kazer SW, Gaillard A, Kolb KE, Villani AC, Johannessen CM, Andreev AY, Van Allen EM, Bertagnolli M, Sorger PK, Sullivan RJ, Flaherty KT, Frederick DT, Jane-Valbuena J, Yoon CH, Rozenblatt-Rosen O, Shalek AK, Regev A, Garraway LA (2016) Dissecting the multicellular ecosystem of metastatic melanoma by single-cell RNA-seq. Science 352(6282):189–196. https://doi.org/10.1126/science.aad0501
Plitas G, Konopacki C, Wu K, Bos PD, Morrow M, Putintseva EV, Chudakov DM, Rudensky AY (2016) Regulatory T cells exhibit distinct features in human breast cancer. Immunity 45(5):1122–1134. https://doi.org/10.1016/j.immuni.2016.10.032
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De Simone, M., Rossetti, G., Pagani, M. (2019). Chromium 10× Single-Cell 3′ mRNA Sequencing of Tumor-Infiltrating Lymphocytes. 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_7
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DOI: https://doi.org/10.1007/978-1-4939-9240-9_7
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Publisher Name: Humana, New York, NY
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