Seq-Well: A Sample-Efficient, Portable Picowell Platform for Massively Parallel Single-Cell RNA Sequencing

Tp, Aicher; Carroll, Shaina L.; Raddi, Gianmarco; Gierahn, Todd M.; Mh, Wadsworth; Tk, Hughes; Love, Clare J.; Ak, Shalek

doi:10.1007/978-1-4939-9240-9_8

Cited by 39 publications

(36 citation statements)

References 13 publications

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“…Organoids were dissociated, and populations of calcein NVOC+ cells and background (i.e., unphotoactivated) cells were isolated by FACS. Subsequent scRNA-seq using the Seq-Well platform [18][19][20] revealed three cell states within this organoid model ( Fig. 2c, Supplementary Table 1), and SPACECAT enabled us to map cells to their microanatomical origin ( Fig.…”

Section: Precise Cell Tagging Of Human Intestinal Organoid Morphologimentioning

confidence: 99%

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Spatially-Resolved Live Cell Tagging and Isolation Using Protected Photoactivatable Cell Dyes

Genshaft¹,

Ziegler²,

Tzouanas³

et al. 2020

Preprint

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Whether cultured in vitro or part of a complex tissue in vivo, a cell's phenotype and function are significantly influenced by dynamic interactions with its microenvironment. To explicitly examine how a cell's spatiotemporal activity impacts its behavior, we developed and validated a strategy termed SPACECAT-Spatially PhotoActivatable Color Encoded Cell Address Tags-to annotate, track, and isolate specific cells in a non-destructive, viability-preserving manner. In SPACECAT, a biological sample is immersed in a photocaged fluorescent molecule, and cells within a location of interest are labeled for further study by uncaging that molecule with user-patterned near-UV 10 light. SPACECAT offers high spatial precision and temporal stability across diverse cell and tissue types, and is compatible with common downstream assays, including flow cytometry and singlecell RNA-Seq. Illustratively, we leveraged this approach in patient-derived intestinal organoids, a spatially complex system less amenable to genetic manipulations, to select for crypt-like regions enriched in stem-like and actively mitotic cells. Moreover, we demonstrate its applicability and utility on ex vivo tissue sections from four healthy organs and an autochthonous lung tumor model, uncovering spatially-biased gene expression patterns among immune cell subsets and identifying rare myeloid phenotypes enriched around tumor/healthy border regions. In sum, our method provides a minimally invasive and broadly applicable approach to link cellular spatiotemporal features and/or behavioral phenotypes with diverse downstream assays, enabling fundamental 20 insights into the connections between tissue microenvironments and biological (dys)function.

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Section: Precise Cell Tagging Of Human Intestinal Organoid Morphologimentioning

confidence: 99%

“…Seq-Well was performed as previously described with changes noted below [18][19][20] . Briefly, 650 a functionalized PDMS nanowell array was loaded with uniquely-barcoded mRNA capture beads (ChemGenes) and suspended in complete media for at least 20 minutes.…”

Section: Generation Of Scrna-seq Libraries Using Seq-wellmentioning

confidence: 99%

Spatially-Resolved Live Cell Tagging and Isolation Using Protected Photoactivatable Cell Dyes

Genshaft¹,

Ziegler²,

Tzouanas³

et al. 2020

Preprint

Self Cite

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“…Other studies have modified and improved the scRNA-seq protocol. The advanced methods include Smart-seq [ 10 , 11 ], CEL-seq [ 12 , 13 ], Qualtz-seq [ 14 ], MARS-seq [ 15 ], Cyto-seq [ 16 ], SUPeR-seq [ 17 ], Drop-seq [ 18 ], InDrop [ 19 ], MATQ-seq [ 20 ], Chromium [ 21 ], sci-RNA-seq [ 22 ], Seq-Well [ 23 ], DroNC-seq [ 24 ], and SPLiT-seq [ 25 ] ( Table 1 ). Generally, scRNA-seq involves the following steps: preparation of in vitro or in vivo samples, dissociation of the sample into single cells, barcode tagmentation of individual cells and reverse transcription, library preparation, massively parallel sequencing, and downstream bioinformatics analysis ( Figure 1 ).…”

Section: Evolution Of Scrna-seq Techniquementioning

confidence: 99%

Insights from the Applications of Single-Cell Transcriptomic Analysis in Germ Cell Development and Reproductive Medicine

Yoo

Thang

et al. 2021

IJMS

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Mechanistic understanding of germ cell formation at a genome-scale level can aid in developing novel therapeutic strategies for infertility. Germ cell formation is a complex process that is regulated by various mechanisms, including epigenetic regulation, germ cell-specific gene transcription, and meiosis. Gonads contain a limited number of germ cells at various stages of differentiation. Hence, genome-scale analysis of germ cells at the single-cell level is challenging. Conventional genome-scale approaches cannot delineate the landscape of genomic, transcriptomic, and epigenomic diversity or heterogeneity in the differentiating germ cells of gonads. Recent advances in single-cell genomic techniques along with single-cell isolation methods, such as microfluidics and fluorescence-activated cell sorting, have helped elucidate the mechanisms underlying germ cell development and reproductive disorders in humans. In this review, the history of single-cell transcriptomic analysis and their technical advantages over the conventional methods have been discussed. Additionally, recent applications of single-cell transcriptomic analysis for analyzing germ cells have been summarized.

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“…To complete this effort, we first needed to realize a map of all LN cell subsets at the molecular level. We therefore generated a singlecell transcriptomic atlas of steady-state murine inguinal LNs (n=7) using the Seq-Well platform (Aicher et al, 2019;Gierahn et al, 2017) (Table S3, see Methods).…”

Section: Scrna-seq Of Ln Cells Nominates Interacting Partners Of Ln-imentioning

confidence: 99%

“…Single cells from each lymph node prior to and post CD3e and CD19 depletion were kept separate and diluted to 15,000 cells in 200 µL complete media (RPMI 1640/10% FBS). Seq-Well was performed as described with changes noted below (Aicher et al, 2019;Gierahn et al, 2017). Briefly, a pre-functionalized PDMS array containing ~86,000 nanowells was loaded with uniquely-barcoded mRNA capture beads (ChemGenes) (Macosko et al, 2015) and suspended in complete media for at least 20 minutes.…”

Section: Ln Scrna-seq Using Seq-wellmentioning

confidence: 99%

Sensory Neurons Innervate Peripheral Lymph Nodes and Locally Regulate Gene Expression in Postsynaptic Endothelium, Stromal Cells, and Innate Leukocytes

Huang

Ziegler

Austin

et al. 2019

Preprint

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Immune responses within barrier tissues are regulated, in part, by nociceptors, specialized peripheral sensory neurons that detect noxious stimuli. Previous work has shown that nociceptor ablation not only alters local responses to immune challenge at peripheral sites, but also within draining lymph nodes (LNs). The mechanisms and significance of nociceptor-dependent modulation of LN function are unknown. Indeed, although sympathetic innervation of LNs is well documented, it has been unclear whether the LN parenchyma itself is innervated by sensory neurons. Here, using a combination of high-resolution imaging, retrograde viral tracing, single-cell 30 transcriptomics (scRNA-seq), and optogenetics, we identified and functionally tested a sensory neuro-immune circuit that is preferentially located in the outermost cortex of skin-draining LNs. Transcriptomic profiling revealed that there are at least four discrete subsets of sensory neurons that innervate LNs with a predominance of peptidergic nociceptors, and an innervation pattern that is distinct from that in the surrounding skin. To uncover potential LN-resident communication partners for LN-innervating sensory neurons, we employed scRNA-seq to generate a draft atlas of all murine LN cells and, based on receptor-ligand expression patterns, nominated candidate target populations among stromal and immune cells. Using selective optogenetic stimulation of LN-innervating sensory axons, we directly experimentally tested our inferred connections. Acute neuronal activation triggered rapid transcriptional changes preferentially within our top-ranked putative interacting partners, principally endothelium and other nodal stroma cells, as well as several innate leukocyte populations. Thus, LNs are monitored by a unique 40 population of sensory neurons that possesses immunomodulatory potential. 840 and Harvard. J.O

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Seq-Well: A Sample-Efficient, Portable Picowell Platform for Massively Parallel Single-Cell RNA Sequencing

Cited by 39 publications

References 13 publications

Spatially-Resolved Live Cell Tagging and Isolation Using Protected Photoactivatable Cell Dyes

Spatially-Resolved Live Cell Tagging and Isolation Using Protected Photoactivatable Cell Dyes

Insights from the Applications of Single-Cell Transcriptomic Analysis in Germ Cell Development and Reproductive Medicine

Sensory Neurons Innervate Peripheral Lymph Nodes and Locally Regulate Gene Expression in Postsynaptic Endothelium, Stromal Cells, and Innate Leukocytes

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