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

项目总结/摘要 BRAIN倡议提出了神经元细胞类型普查的发展，作为实现 绘制神经元回路的结构和组件。有几个研究小组使用单细胞RNA 测序（scRNA-seq）以显示RNA转录物水平是细胞类型特异性的，并且通过分离和 通过对单个细胞的RNA进行测序，他们可以创建大脑中细胞类型的目录。同样地， 单核甲基胞嘧啶测序（snmC-seq）显示了DNA甲基化（mC）模式， 基因组是高度细胞类型特异性，也可用于确定细胞身份。全脑普查 在目前的努力下，大脑内细胞类型空间映射的技术开发已经成为一种 主要焦点。几个研究小组已经显示了利用scRNA-seq数据和RNA测序技术的潜力。 荧光原位杂交（RNA FISH）或原位测序，以创建细胞中细胞类型的空间图 培养物和组织切片，尽管还没有这样的努力被报道用于使用 snmC-seq数据。因此，这项提案将寻求测试和开发一种利用细胞类型的方法- 特定的snmC-seq数据正在为小鼠大脑图谱生成，以开发这些细胞的3D图谱 在小鼠的额叶皮层中。荧光原位测序（FISSEQ）已用于测序 RNA分子的碱基分辨率，也可以用于DNA原位测序，但这种方法还没有 可适用于原位甲基胞嘧啶测序。此外，这种方法受到包括两个 限制其效率的步骤产生了可以容易测序的分子。第一，就地 使用FISSEQ进行测序需要将RNA逆转录成cDNA，这一技术的创造者 已经确定这种逆转录是限制其效率的主要因素。因此，细胞分型 其中不需要逆转录策略应该固有地更有效。二是 FISSEQ方案要求通过分子内连接产生环状cDNA，但不能形成环状cDNA。 末端到末端的环在能量上是非常不利的，因为原位测序需要DNA的交联， 固定在细胞内的蛋白质。该提案要求开发一种方法，其中DNA 化学转化用于甲基胞嘧啶原位测序，通过发夹直接连接环化 衔接子与平端DNA连接，然后进行靶向原位测序。该项目的成功完成将导致 在一种通过甲基胞嘧啶模式空间映射细胞类型的方法中， 通过逆转录和分子内连接引入。这种技术称为原位甲基胞嘧啶 测序（MIS-SEQ）将与组织清除技术和光片荧光显微镜技术配对 来对厚的老鼠大脑切片进行测序。这些技术的结合将允许在原位 来自6个切片的整个小鼠额叶皮质中的细胞类型的甲基胞嘧啶测序和空间映射 （约430 μm切片）。

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. The combination of these techniques will allow in-situ methylcytosine sequencing and spatial mapping of cell types in the entire mouse frontal cortex from 6 slices (~430 μm sections).