Abstract
Single-cell functional analysis provides a natural next step in the now widely adopted single-cell mRNA sequencing studies. Functional studies can be designed to study cellular context by using single-cell culture, perturbation, manipulation, or treatment. Here we present a method for a functional study of 48 single cells by single-cell isolation, dosing, and mRNA sequencing with an integrated fluidic circuit (IFC) on the Fluidigm® Polaris™ system. The major procedures required to execute this protocol are (1) cell preparation and staining; (2) priming, single-cell selection, cell dosing, cell staining, and cDNA generation on the Polaris IFC; and (3) preparation and sequencing of single-cell mRNA-seq libraries. The cell preparation and staining steps employ the use of a universal tracking dye to trace all cells that enter the IFC, while additional fluorescence dyes chosen by the user can be used to differentiate cell types in the overall mix. The steps on the Polaris IFC follow standard protocols, which are also described in the Fluidigm user documentation. The library preparation step adds Illumina® Nextera® XT indexes to the cDNA generated on the Polaris IFC. The resulting sequencing libraries can be sequenced on any Illumina sequencing platform.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Guo G, Pinello L, Han X et al (2016) Single human T cells stimulated in the absence of feeder cells transcribe interleukin-2 and undergo long-term clonal growth in response to defined monoclonal antibodies and cytokine stimulation. Cell Rep 14:956–965. https://doi.org/10.1016/j.celrep.2015.12.089
Kimmerling RJ, Lee Szeto G, Li JW et al (2016) A microfluidic platform enabling single-cell RNA-seq of multigenerational lineages. Nat Commun 7:10220. https://doi.org/10.1038/ncomms10220
Sunder-Plassmann R, Breiteneder H, Zimmermann K et al (1996) Single human T cells stimulated in the absence of feeder cells transcribe interleukin-2 and undergo long-term clonal growth in response to defined monoclonal antibodies and cytokine stimulation. Blood 87(12):5179–5184
Prakadan SM, Shalek AK, Weitz DA (2017) Scaling by shrinking: empowering single-cell 'omics' with microfluidic devices. Nat Rev Genet 18(6):345–361. https://doi.org/10.1038/nrg.2017.15
Dura B, Dougan SK, Barisa M et al (2015) Profiling lymphocyte interactions at the single-cell level by microfluidic cell pairing. Nat Commun 6:5940. https://doi.org/10.1038/ncomms6940
Etzrodt M, Schroeder T (2017) Illuminating stem cell transcription factor dynamics: long-term single-cell imaging of fluorescent protein fusions. Curr Opin Cell Biol 49:77–83. https://doi.org/10.1016/j.ceb.2017.12.006
Ramalingam N, Fowler B, Szpankowski L et al (2016) Fluidic logic used in a systems approach to enable integrated single-cell functional analysis. Front Bioeng Biotechnol 4:70. https://doi.org/10.3389/fbioe.2016.00070
Wills QF, Mellado-Gomez E, Nolan R et al (2017) The nature and nurture of cell heterogeneity: accounting for macrophage gene-environment interactions with single-cell RNA-Seq. BMC Genomics 18(1):53
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2019 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Sanada, C.D., Ooi, A.T. (2019). Single-Cell Dosing and mRNA Sequencing of Suspension and Adherent Cells Using the PolarisTM System. 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_12
Download citation
DOI: https://doi.org/10.1007/978-1-4939-9240-9_12
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-4939-9239-3
Online ISBN: 978-1-4939-9240-9
eBook Packages: Springer Protocols