Probe-Seq enables transcriptional profiling of specific cell types from heterogeneous tissue by RNA-based isolation

Abstract

Recent transcriptional profiling technologies are uncovering previously-undefined cell populations and molecular markers at an unprecedented pace. While single cell RNA (scRNA) sequencing is an attractive approach for unbiased transcriptional profiling of all cell types, a complementary method to isolate and sequence specific cell populations from heterogeneous tissue remains challenging. Here, we developed Probe-Seq, which allows deep transcriptional profiling of specific cell types isolated using RNA as the defining feature. Dissociated cells are labeled using fluorescent in situ hybridization (FISH) for RNA, and then isolated by fluorescent activated cell sorting (FACS). We used Probe-Seq to purify and profile specific cell types from mouse, human, and chick retinas, as well as from Drosophila midguts. Probe-Seq is compatible with frozen nuclei, making cell types within archival tissue immediately accessible. As it can be multiplexed, combinations of markers can be used to create specificity. Multiplexing also allows for the isolation of multiple cell types from one cell preparation. Probe-Seq should enable RNA profiling of specific cell types from any organism.

Multiplexed detection and isolation of viable low-frequency cytokine-secreting human B cells using cytokine secretion assay and flow cytometry (CSA-Flow)

“…The assay relies on sequential antibody-based cell surface labelling, thereby preserving cell integrity and minimizing cell loss 34,35 . These features, along with the ability to isolate viable and functional low frequency cytokine-defined cells, encapsulate the main competitive advantage of this assay over traditional intracellular cytokine-staining or flow fluorescent in situ hybridization [19][20][21][22][23][24][25] . Assay multiplexing is attractive for maximizing the output of potentially limited clinical sample volumes, and has been previously used with CSA to both isolate and distinguish distinct cytokine-secreting immune cells 42,43 .…”

“…Indeed, the isolation of highly purified and viable cytokine-defined immune cells is essential for robust interrogation of individual cells such as by scRNA-Seq 17 , and is also required for other downstream applications such as the assessment of the impact of such cells on responses of other cells in vitro, or when introduced in vivo (potentially in the form of adoptive cell transfer 18 ). However, the isolation of viable and purified cytokine-expressing immune cells has presented a technical challenge, as commonly used intracellular cytokine-staining (ICS) or flow-fluorescent in-situ hybridization approaches involve cell fixation and permeabilization 19 – 25 . The resultant loss of cell viability and integrity seriously limits the use of cells for comprehensive functional profiling and downstream applications 19 – 25 .…”

“…impact of such cells on responses of other cells in vitro, or when introduced in vivo (potentially in the form of adoptive cell transfer 18 ). However, the isolation of viable and purified cytokine-expressing immune cells has presented a technical challenge, as commonly used intracellular cytokine-staining (ICS) or flow-fluorescent in-situ hybridization approaches involve cell fixation and permeabilization [19][20][21][22][23][24][25] . The resultant loss of cell viability and integrity seriously limits the use of cells for comprehensive functional profiling and downstream applications [19][20][21][22][23][24][25] .…”

“…However, the isolation of viable and purified cytokine-expressing immune cells has presented a technical challenge, as commonly used intracellular cytokine-staining (ICS) or flow-fluorescent in-situ hybridization approaches involve cell fixation and permeabilization [19][20][21][22][23][24][25] . The resultant loss of cell viability and integrity seriously limits the use of cells for comprehensive functional profiling and downstream applications [19][20][21][22][23][24][25] . While viable cells can be sorted based on certain cell surface-markers that have been reported to enrich for particular cytokine-expressing immune cells, such markers are inevitably neither fully sensitive nor specific [26][27][28][29][30][31][32][33] .…”

Extraction of nuclei from archived post-mortem tissues for single-nucleus sequencing applications

“…High-throughput in situ hybridization (ISH) 28 and ISH based nuclei sorting 27 have also been used to confirm experimentally determined cell-types from snRNA-seq. In the case of complex tissues, it can be useful to perform careful dissection and even to cryosection the tissue before preparing nuclei to ensure that the approximate cell-type composition for each sample will be comparable 11,24 .…”