Continuing Development of Find-Seq for Gene Expression Analysis of Rare Cell Populations

Authors

  • P Rezvan Sangsari
  • N Dulin
  • P Ramdas
  • J Krynitsky
  • EA Boritz
  • NY Morgan

Abstract

Gene expression analysis of rare cell populations is a powerful tool for a range of problems in biomedical research, but identifying and isolating these cells is a significant challenge when the cells lack surface markers – for example, HIV-infected CD4+ T cells in individuals on antiretroviral therapy. This work advances microfluidic technologies to improve the isolation and transcriptomic analysis of these HIV-infected cells, building on prior work by Clark et al. (2023). The FIND-seq method starts with simultaneous lysis, encapsulation, and transcriptome capture of single cells in agarose microbeads covalently linked with poly(T) oligonucleotides, followed by batch reverse transcription. The agarose beads are subsequently re-encapsulated with a PCR mix, enabling whole transcriptome amplification and fluorescence-based sorting of HIV-DNA+ beads using an HIV-gag TaqMan probe. In this work that is now being established in the IRP, we have substantially modified agarose bead conjugation, purification, storage, and the initial encapsulation step to increase the retention of genomic DNA and RNA, achieving improved binding capabilities and reduced device clogging. With optimized conditions, the 55 um diameter agarose beads showed a capture capacity of approximately 2 pg RNA, maintaining high transcriptome quality. Furthermore, we implemented a custom dielectrophoretic microfluidic sorter with fluorescence detection for efficient bead sorting, employing real-time signal monitoring and targeted deflection using LabVIEW. Overall, this work facilitates the targeted study of rare cell populations, advancing potential therapeutic strategies against HIV.

Scientific Focus Area: Biomedical Engineering and Biophysics

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