# Spatially Resolved Methylomes to Map Neuronal Cell-Type Connectivity in Tissue

> **NIH NIH F32** · SALK INSTITUTE FOR BIOLOGICAL STUDIES · 2020 · $54,790

## Abstract

Project Summary/Abstract
The BRAIN initiative has put forth the development of a neuronal cell-type census as the first step towards
mapping the structure and components of neuronal circuits. Several groups have used single-cell RNA
sequencing (scRNA-seq) to shown that RNA transcript levels are cell type-specific, and by isolating and
sequencing the RNA from individual cells they can create a catalogue of cell types within the brain. Likewise,
single-nucleus methylcytosine sequencing (snmC-seq) has shown DNA methylation (mC) patterns within the
genome are highly cell type-specific and may also be used to define cellular identity. With whole-brain census
efforts underway, technology development for spatial mapping of cell types within the brain has become a
major focus. Several groups have shown the potential of techniques which leverage scRNA-seq data and RNA
fluorescent in-situ hybridization (RNA FISH) or in-situ sequencing to create spatial maps of cell types in cell
cultures and tissue sections, though no such efforts have been reported for spatial mapping of cell types using
snmC-seq data. Therefore, this proposal will seek to test and develop a method that leverages the cell type-
specific snmC-seq data that is being generated for the mouse brain atlas to develop a 3D map of these cell
types within the frontal cortex of mice. Fluorescent in-situ sequencing (FISSEQ) has been used to sequence
RNA molecules at base resolution and can also be used to sequence DNA in-situ, but this method has yet to
be adapted for in-situ methylcytosine sequencing. In addition, this method is hampered by the inclusion of two
steps that limit the efficiency with it creates molecules that can be readily sequenced. Firstly, in-situ
sequencing using FISSEQ calls for the RNA to be reverse transcribed into cDNA, the creators of this technique
have identified this reverse transcription as a major factor limiting it’s efficiency. Therefore, a cell-typing
strategy wherein no reverse transcription is necessary should be inherently more efficient. Secondly, the
FISSEQ protocol calls for the production of circular cDNA through intramolecular ligation, but the formation of
end-to-end loops is highly energetically unfavorable, as in-situ sequencing requires crosslinking of DNA and
proteins within the cell to fix them in place. This proposal calls for the development of a method wherein DNA
is chemically converted for methylcytosine sequencing in-situ, circularized through the direct ligation of hairpin
adaptors to blunt DNA, followed by targeted in-situ sequencing. Successful completion of this project will result
in a method to spatially map cell-types by their methyl-cytosine patterns, bypassing the inefficiencies
introduced by reverse transcription and intramolecular ligation. This technique called methyl-cytosine in situ
sequencing (MIS-SEQ) will be paired with tissue clearing techniques and light sheet fluorescence microscopy
in order to sequence thick mouse brain slices....

## Key facts

- **NIH application ID:** 9998746
- **Project number:** 5F32MH118706-03
- **Recipient organization:** SALK INSTITUTE FOR BIOLOGICAL STUDIES
- **Principal Investigator:** Robert Youssefian Henley
- **Activity code:** F32 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $54,790
- **Award type:** 5
- **Project period:** 2018-09-14 → 2021-05-28

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9998746

## Citation

> US National Institutes of Health, RePORTER application 9998746, Spatially Resolved Methylomes to Map Neuronal Cell-Type Connectivity in Tissue (5F32MH118706-03). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/9998746. Licensed CC0.

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