Poster Y18 - Continuing Development of Find-Seq for Gene Expression Analysis of Rare Cell Populations

Introduction: The isolation of rare cells that lack surface markers for subsequent gene expression analysis poses a considerable challenge for many research questions. One example of this is the isolation of CD4+ T cells harboring HIV in people on antiretroviral therapy. Understanding the unique characteristics of this cell population, which is only identifiable by looking for HIV DNA using PCR, could greatly assist efforts to develop targeted therapies and someday find a full cure for the viral infection. To address this challenge, we build on proof-of-concept work using microfluidic technologies to isolate and analyze the transcriptomes of HIV-infected cells based on HIV DNA detection (Clark, 2023), making several improvements to the workflow to enhance efficiency and ease-of-use. By making the experimental approach more accessible, we hope to broaden the use of the technique in biomedical research laboratories and to enable more thorough validation of the sequencing data.

Materials and

Methods: A series of microfluidic systems were used for the unbiased detection and gene expression analysis of HIV-infected cells directly from patient samples, building on methods previously developed for focused interrogation of cells by nucleic acid detection and sequencing (FIND-seq). The initial step of FIND-seq is simultaneous lysis, encapsulation, and transcriptome capture in microbeads of agarose covalently conjugated with poly(T) oligonucleotides. In this work, we present modifications to the conjugation, purification, and storage of the functionalized agarose in order to improve selective binding and retention of genomic DNA and polyadenylated RNA. After encapsulation and RNA capture, the whole-transcriptome cDNA is covalently linked to the hydrogel via batch reverse transcription in the agarose beads. A second microfluidic device is then used to encapsulate the agarose beads with a PCR mix that includes a HIV-gag TaqMan probe, enabling whole transcriptome amplification and fluorescence-based sorting of HIV-DNA+ beads. At present, this sorting step is achieved with a third custom microfluidic device, but part of this work explores the possibility of expediting the sorting process, for example by incorporating a FACS presort for cell-occupied beads prior to PCR.

Results, Conclusions, and Discussions: We present the results of validation studies using cell lines that show significant improvement in binding and retention of the transcriptomic material within agarose beads after modification of the conjugation reaction protocol and adaptation of electrophoretic rinsing to remove unbound primer. During encapsulation, we implemented a simple custom sheath heater between the agarose syringe and the device which has virtually eliminated device clogging.

In a series of measurements using soluble RNA in lieu of cells, and poly(T)-conjugated agarose, diluted approximately 20-fold in unmodified agarose to a primer concentration of 11.2 ng/ul, we found a capture capacity of roughly 2 pg RNA per 55um diameter bead. Electrophoretic rinsing to remove unbound primer does not significantly affect capture efficiency of the primer while significantly reducing processing time. Bioanalyzer traces further confirm the quality of the captured transcriptome in the beads for both soluble RNA and capture from a J-Lat cell line.



For sorting, we have implemented a version of the previously published dielectrophoretic microfluidic sorter integrated with fluorescence detection (Clark, 2018). The microscope-based platform enables simultaneous imaging of the sorter device and wavelength-specific detection of fluorescence from beads passing through the detection zone using a photomultiplier tube (PMT) for speed and sensitivity. The PMT signal is sent to an analog input on a field-programmable gate array (FPGA) board, which is controlled by a LabVIEW program that monitors the signal in real-time and triggers a series of output pulses whenever a positive bead is detected. These output pulses are fed through a high-voltage amplifier and generate DEP-based deflection of the selected beads into the collection channel. We will present our experience optimizing this system as well as efforts to leverage commercial instruments to increase throughput and efficiency.

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