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Single-cell RNA Sequencing of Fluorescently Labeled Mouse Neurons Using Manual Sorting and Double In Vitro Transcription with Absolute Counts Sequencing DIVA-Seq
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Automated Laser-Assisted Single-Cell Sorting for Cell Functional and RNA Sequencing.

Yuntong Wang1,2,3,4, Ying Xue5, Huan Wang1,2

  • 1Changchun Institute of Optics, Fine Mechanics and Physics, Chinese Academy of Sciences, Changchun 130033, P. R. China.

ACS Sensors
|January 22, 2025
PubMed
Summary

This study introduces an automated single-cell sorter using laser-induced forward transfer (LIFT) and micropore arrays for high-efficiency cell isolation. This advanced technology ensures precise sorting and high cell viability for downstream single-cell analysis.

Keywords:
RNA sequencinglaser-induced forward transfermicroporessingle-cell analysissingle-cell sorting

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Area of Science:

  • Biotechnology
  • Cell Biology
  • Genomics

Background:

  • Accurate single-cell sorting is vital for analyzing cellular heterogeneity and function.
  • Conventional methods like manual micromanipulation and fluorescence-activated cell sorting have limitations in throughput, efficiency, precision, and cell viability.
  • Current single-cell analysis demands more advanced sorting technologies.

Purpose of the Study:

  • To develop and validate an automated, highly efficient single-cell sorter.
  • To improve single-cell isolation efficiency, precision, and cell viability for downstream applications.
  • To enable reliable single-cell RNA sequencing and multiomics analysis.

Main Methods:

  • Integration of laser-induced forward transfer (LIFT) with a high-throughput picoliter micropore array.
  • Surface functionalization of the micropore array to control liquid surface tension and form single-cell droplets.
  • Development of an in-house microscopic system for automated identification and retrieval of rare target cells using LIFT.
  • Demonstration of sorting transfected PC-9 cells and subsequent cell culture.

Main Results:

  • Achieved automated and highly efficient single-cell isolation (>80%) using the micropore array.
  • Demonstrated precise sorting efficiency (approx. 100%) and high cell viability (approx. 80%) for rare target cells via LIFT.
  • Successfully performed single-cell RNA sequencing, obtaining high-quality transcriptome data without additional costs due to sorting inaccuracies.
  • Minimized cell loss and contamination risk during the sorting of rare transfected cells.

Conclusions:

  • The developed automated single-cell sorter significantly overcomes limitations of conventional methods.
  • This technology enables efficient and reliable isolation of single cells, crucial for advancing single-cell analysis and multiomics.
  • The sorter provides a foundation for precision medicine research by facilitating accurate cellular analysis.