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Related Experiment Video

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Microfluidics-based High-throughput Circulating Tumor Cell Sorting and Single-cell Sequencing Technology
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Optimization of single-cell plate sorting for high throughput sequencing applications.

Lauren E Higdon1, Corey J Cain2, Melissa A Colden1

  • 1Stanford University, Department of Medicine/Nephrology, Palo Alto, CA 94304, United States.

Journal of Immunological Methods
|December 28, 2018
PubMed
Summary

Optimizing flow cytometry sorting parameters, like nozzle size and flow rates, improves single-cell sequencing yields. This enables accurate linked protein and gene expression analysis, crucial for studying rare immune cells.

Keywords:
Flow rateIndex sortNozzlePlate sortSingle cell sequencingThreshold rate

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Area of Science:

  • Immunology
  • Genomics
  • Cell Biology

Background:

  • Single-cell sequencing is increasingly used in immunology.
  • Flow cytometry index sorting links protein data to sequences but requires parameter optimization.

Purpose of the Study:

  • To present considerations and optimization data for flow cytometric index sorting parameters.
  • To improve cell yields and sequencing read counts for single-cell analysis.

Main Methods:

  • Optimization of nozzle size, flow rate, and threshold rate for index sorting.
  • Analysis of linked protein and gene expression for interferon-gamma (IFNγ).

Main Results:

  • Minimizing flow rates and using a 70 µm nozzle enhanced cell yields.
  • Decreasing the threshold rate improved total sequencing read counts.
  • Demonstrated correlation between IFNγ protein expression and IFNG mRNA levels on a single-cell basis.

Conclusions:

  • Rigorous optimization of plate sorting parameters is essential for successful single-cell sequencing.
  • Recommended parameters include a 70 µm nozzle and low flow/threshold rates for rare human lymphocyte analysis.