See-N-Seq: RNA sequencing of target single cells identified by microscopy via micropatterning of hydrogel porosity

Author:

Lee Jeong Hyun,Park Emily S.ORCID,Choi Jane RuORCID,Matthews Kerryn,Lam Alice V.,Deng Xiaoyan,Duffy Simon P.,Ma HongshenORCID

Abstract

AbstractSingle cell RNA sequencing has the potential to elucidate transcriptional programs underlying key cellular phenotypes and behaviors. However, many cell phenotypes are incompatible with indiscriminate single cell sequencing because they are rare, transient, or can only be identified by imaging. Existing methods for isolating cells based on imaging for single cell sequencing are technically challenging, time-consuming, and prone to loss because of the need to physically transport single cells. Here, we developed See-N-Seq, a method to rapidly screen cells in microwell plates in order to isolate RNA from specific single cells without needing to physically extract each cell. Our approach involves encapsulating the cell sample in a micropatterned hydrogel with spatially varying porosity to selectively expose specific cells for targeted RNA extraction. Extracted RNA can then be captured, barcoded, reverse transcribed, amplified, and sequenced at high-depth. We used See-N-Seq to isolate and sequence RNA from cell-cell conjugates forming an immunological synapse between T-cells and antigen presenting cells. In the hours after synapsing, we found time-dependent bifurcation of single cell transcriptomic profiles towards Type 1 and Type 2 helper T-cells lineages. Our results demonstrate how See-N-Seq can be used to associate transcriptomic data with specific functions and behaviors in single cells.

Funder

University of British Columbia

Mitacs

Michael Smith Foundation for Health Research

Gouvernement du Canada | Canadian Institutes of Health Research

Gouvernement du Canada | Natural Sciences and Engineering Research Council of Canada

Prostate Cancer Canada

Publisher

Springer Science and Business Media LLC

Subject

General Agricultural and Biological Sciences,General Biochemistry, Genetics and Molecular Biology,Medicine (miscellaneous)

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