# High-throughput single-cell co-sequencing of small and large RNAs to identify molecular circuitry in cancer

> **NIH NIH R33** · YALE UNIVERSITY · 2020 · $417,077

## Abstract

PROJECT SUMMARY
Small RNAs (smRNAs) are important non-coding regulators broadly implicated in human cancer initiation and
progression. The functions and mechanism of smRNAs have been widely studied in cancer research. They are
also being investigated as new therapeutic targets but the results so far are unsatisfactory, due in part to the
complexity of miRNA-mRNA regulatory network coupled with cellular heterogeneity in human cancers. Thus,
new tools that can co-profile smRNAs and large RNAs (lgRNAs) in large numbers of single cells can help
address this long-standing challenge, add a new dimension to smRNA research, empower the discovery of
new mechanisms of smRNAs to participate in cancer biology, and enable potential applications to cancer
diagnostics and therapeutics. A close collaboration between PIs Fan and Lu has demonstrated, for the first
time, co-sequencing of both smRNAs and lgRNAs from the same single cells (Wang et al., Nature Comm.,
2019). It further showed that having paired smRNA and lgRNA profiles on the single-cell level can reveal
miRNA-mediated gene regulation and new mechanisms for controlling miRNA expression and intercellular
heterogeneity. However, this single-cell smRNA-lgRNA co-sequencing technology is a manual process with
low throughput and high cost, limiting its potential for cancer research due to insufficient statistic power to
interrogate highly heterogeneous tumor cells. In this application, we propose the advanced development of this
technology to deliver a high-throughput single-cell smRNA/lgRNA co-sequencing (scSLRco-seq) technology. It
employs a novel slip-transfer microdevice in combination with novel molecular barcoding scheme and
downstream biochemistry for simultaneous analysis of smRNAs and lgRNAs in 1000’s of single cells.
Specifically, we will (Aim 1) develop a microfluidic cross-flow patterning method to create 2,500 DNA barcode
arrays for spatially-coupled capture and barcoding of small and large RNAs, (Aim2) develop a novel slip-
transfer chip to integrate multi-step biochemistry workflow for high throughput scSLRCo-seq, and (Aim3)
validate this technology using human and mouse myeloid leukemia models. This novel technology addresses
the lack of capability for high-throughput single-cell smRNA/lgRNA co-profiling, filling a gap in single-cell omics
field and enabling the study of new questions previously impossible to answer. It represents a major leap in the
field and will find wide-spread use in cancer research and applications.

## Key facts

- **NIH application ID:** 9898733
- **Project number:** 1R33CA246711-01
- **Recipient organization:** YALE UNIVERSITY
- **Principal Investigator:** Rong Fan
- **Activity code:** R33 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $417,077
- **Award type:** 1
- **Project period:** 2020-03-01 → 2023-02-28

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9898733

## Citation

> US National Institutes of Health, RePORTER application 9898733, High-throughput single-cell co-sequencing of small and large RNAs to identify molecular circuitry in cancer (1R33CA246711-01). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/9898733. Licensed CC0.

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