# Single-cell in situ analysis of RNA modifications in intact tissues

> **NIH NIH DP2** · BROAD INSTITUTE, INC. · 2021 · $1,404,000

## Abstract

PROJECT SUMMARY
Cellular RNAs have more than 100 different kinds of chemical modifications, which together comprise the
epitranscriptome. Previous studies have revealed that RNA-modifying pathways are vital to higher eukaryotes,
and the alteration of these pathways is associated with a number of human diseases. However, it is still unclear
why we need such a complex chemical repertoire for RNA and how RNA modifications impact gene expression
in biological tissues with diverse cell types. The major bottleneck for investigating RNA modifications has been
the lack of measurement tools with subcellular and single-cell resolutions. Bulk epitranscriptomic sequencing
methods can only measure the averaged distribution of modification sites from millions of cells, obscuring the
intrinsic heterogeneity of epitranscriptomic states in distinct cell types. Moreover, spatial information is lost during
RNA extraction from tissues, preventing further analysis of RNA modification patterns in the context of tissue
morphology and subcellular compartments. To address such limitations, we propose to create cutting-edge
platforms for three-dimensional (3D) in situ sequencing of RNA modifications. Then we will utilize the new tools
to analyze gene regulation mechanisms mediated by RNA modifications at single-cell resolution in intact
biological tissues. Using the N6-methyladenosine (m6A) pathway in mouse brain tissue as our model system, we
will (a) develop innovative and broadly applicable 3D in situ sequencing methods for RNA modifications; (b)
profile the m6A code at single-cell resolution within intact mouse brain tissue; (c) study how m6A modifications in
various cell types work collectively and interactively to regulate brain function. In total, this proposal will lead to
(1) a transformative toolbox for single-cell epitranscriptomic profiling with broad applications in various biological
tissues, (2) a spatially resolved single-cell RNA modification atlas in the mouse brain, and (3) new scientific
understanding on how RNA modifications impact tissue function. In the long term, we aim to reveal new principles
of post-transcriptional gene regulation at subcellular and single-cell resolutions in complex biological systems,
as well as discover new chemical fingerprints of RNAs in health and disease.

## Key facts

- **NIH application ID:** 10245901
- **Project number:** 1DP2GM146245-01
- **Recipient organization:** BROAD INSTITUTE, INC.
- **Principal Investigator:** Xiao Wang
- **Activity code:** DP2 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $1,404,000
- **Award type:** 1
- **Project period:** 2021-09-23 → 2024-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10245901

## Citation

> US National Institutes of Health, RePORTER application 10245901, Single-cell in situ analysis of RNA modifications in intact tissues (1DP2GM146245-01). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10245901. Licensed CC0.

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