Fluorescence-Activated Nuclei Negative Sorting of Neurons Combined with Single Nuclei RNA Sequencing to Study the Hippocampal Neurogenic Niche

Thomas Kerloch; Tja&#353;a Lepko; Kirill Shkura; Fran&#231;ois Guillemot; S&#233;bastien Gillotin

doi:10.3791/64369

JoVE Journal
Neuroscience

This content is Free Access.

JoVE Journal Neuroscience

Fluorescence-Activated Nuclei Negative Sorting of Neurons Combined with Single Nuclei RNA Sequencing to Study the Hippocampal Neurogenic Niche

荧光激活神经元核负分选结合单核RNA测序研究海马神经源性生态位

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08:16 min

October 20, 2022

DOI: 10.3791/64369-v

Thomas Kerloch¹, Tjaša Lepko², Kirill Shkura², François Guillemot¹, Sébastien Gillotin²

¹The Francis Crick Institute, ²MSD R&D Innovation Centre

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Overview

This study presents a method to sequence single nuclei isolated from the mouse dentate gyrus, utilizing fluorescence-activated nuclei (FAN)-sorting to exclude most neurons. This approach facilitates the examination of various cell types, particularly rare populations like neural stem cells, enhancing our understanding of the adult hippocampal niche.

Key Study Components

Area of Science

Neuroscience
Cell Biology
Molecular Biology

Background

Understanding the cellular composition of the dentate gyrus is crucial for studying neurogenesis.
Traditional approaches often result in the loss of rare cell populations like neural stem cells.
FACS sorting allows for more refined profiling of different cell types.
This method opens avenues for investigating various biological questions related to neurogenesis.

Purpose of Study

To exclude neurons and focus on other cell populations in the dentate gyrus.
To assess the transcriptomic profiles of low-abundance cell types.
To enable adaptable methodologies for various research inquiries.

Methods Used

Single nuclei sequencing and FAN-sorting were applied to isolated nuclei from mouse brain tissue.
The biological model involved neural stem cells, astrocytes, and oligodendrocytes within the dentate gyrus.
No multiomics workflows were specifically mentioned; the focus was on RNA sequencing.
Critical steps include brain dissection, homogenization, and flow cytometry.
Utilization of specific antibodies in FACS gating allows versatility in experimental design.

Main Results

High-quality RNA sequences were achieved, particularly for non-neuronal cell types, which constituted 81.3% of sorted nuclei.
The findings indicated distinct clusters corresponding to known cell types within the dentate gyrus.
Significant transcriptional activity was observed in non-FACS-sorted neurons, emphasizing their prevalence.
This study validates the effectiveness of FACS sorting in preserving nuclei integrity for sequencing.

Conclusions

The method effectively enables the study of diverse cell types in the adult hippocampal niche.
It demonstrates the potential for adapting protocols to investigate different stem cell niches.
Insights from this research can deepen our understanding of neural mechanisms and neurogenesis.

Frequently Asked Questions

What are the advantages of using FAN-sorting?

FAN-sorting allows for the selective exclusion of neurons, enriching the sample for other cell types such as neural stem cells and glial cells, enhancing transcriptomic analysis.

How is the biological model implemented in this study?

The biological model involves dissecting mouse brain tissue to isolate the dentate gyrus, followed by a series of steps to extract and sort nuclei for RNA sequencing.

What types of data do you obtain from this method?

The method provides high-quality RNA sequencing data, revealing the transcriptional profiles of low-abundance cell populations in the dentate gyrus.

How adaptable is this method for other studies?

This protocol is designed to be versatile, as it allows researchers to change antibody targets in FACS gating to investigate various biological questions.

What are the limitations of this approach?

While FAN-sorting enriches for specific cell types, it may still miss some rare populations if not optimized correctly, limiting the comprehensiveness of the analysis.

How does the study contribute to understanding neurogenesis?

By isolating and profiling non-neuronal cell types, the study highlights their significant roles in neuronal modulation and neurogenesis within the adult brain.

这里介绍的是从小鼠齿状回分离的单个细胞核测序的方法，该方法通过荧光激活细胞核（FAN）分选排除大多数神经元。这种方法可生成高质量的表达谱，并促进对生态位中代表的大多数其他细胞类型的研究，包括神经干细胞等稀缺群体。

使用负 FACS 分选排除神经元的策略可产生低丰度细胞群（如神经干细胞、星形胶质细胞和少突胶质细胞）的高质量 RNA 测序速率，以研究它们在成人海马神经发生调节中的作用。使用这种方法，可以调整FACS门控中抗体的选择，这使得该协议在解决与齿状回相关的各种生物学问题方面非常通用。该方法主要用于研究成年海马生态位。

然而，它可以很容易地适应研究其他干细胞生态位。展示该程序的将是弗朗西斯克里克研究所的博士后培训研究员Sara Ahmed de Prado。首先，将从安乐死小鼠解剖的大脑转移到装有冰冷PBS的10厘米培养皿中。

然后将培养皿放在冰上，沿着矢状轴将大脑切成两半。使用手术刀切除小脑。将大脑的一半转移到放置在冰上的新的10厘米培养皿中，其中包含冰冷的PBS。

使用双筒望远镜解剖齿状回或DG，并重复此步骤以从大脑的后半部分获得第二个DG。将两个DG转移到预冷的Dounce均质器中，并加入1毫升冷均质缓冲液或HB。用松散的A杵10笔使组织匀浆，然后用15笔的紧密B杵匀浆。将匀浆转移到预冷的15毫升管中。

用1毫升冷HB冲洗Dounce均质器，并将其与同一管混合。将3毫升HB加入15毫升管中，并在冰上孵育5分钟。通过轻轻倒置试管来混合该细胞核悬浮液两次。

使用0.5毫升HB，预润湿放置在50毫升试管上的70微米过滤器帽，然后在用0.5毫升HB洗涤细胞过滤器之前过滤细胞核悬浮液。接下来，取出细胞过滤器并将试管在 500 x g的旋转桶离心机中以 4 摄氏度离心 5 分钟。弃去上清液。使用 P1000 移液管将沉淀轻轻重悬于 4 毫升 HB 中。

在冰上孵育5分钟后，将悬浮液在500×g下离心10分钟，温度为4摄氏度。弃去上清液并将沉淀重悬于3毫升洗涤介质中。使用0.5毫升洗涤介质在50毫升试管上预润湿35微米过滤器盖。

然后使用 P1000 移液器一次移取 0.5 毫升悬浮液来过滤细胞核悬浮液。用0.5毫升洗涤介质清洗过滤器盖后，将管子放在冰上。将滤液转移到新的15毫升管中，并在500 x g 下离心5分钟和4摄氏度。

弃去上清液，并将沉淀重悬于3毫升洗涤介质中。重复离心并将沉淀重悬于1毫升洗涤介质中，用小鼠抗NeuN，Alexa Fluor 488偶联抗体和每毫升1微克DAPI。在黑暗中在冰上孵育反应45分钟。

对于荧光活化细胞核分选或FANS，将免疫染色的细胞核悬浮液转移到5毫升试管中，并将其放在冰上直到流式细胞术程序开始。将样品以温和的强度涡旋3秒钟，然后将试管放入FACS仪器中。要从染色的细胞核悬浮液中获取数据，请将门设置为DAPI高度和DAPI区域，以排除细胞碎片和聚集的细胞核。

然后在对数侧散射或SSC区域和对数前向散射（FSC）区域中设置门，以将单个细胞核与剩余的DAPI染色聚集体或细胞碎片分开。然后通过设置抗NeuN-AF488和FSC区域的门来隔离NeuN-AF488阴性群体。分析后，使用门控策略在装有 50 微升洗涤介质的 1.5 毫升收集管中对抗 NeuN-AF488 阴性群体进行分类。

分选完成后，向收集管中加入 1 毫升含有 1% 牛血清白蛋白或 BSA 的 PBS，以收集管壁上的液滴，并在 500 x g 下以 4 摄氏度离心管 5 分钟。弃去上清液，留下50微升溶质重悬离心的细胞核。在0.5毫升微管中，将5微升的细胞核悬浮液加入5微升台盼蓝中。

在进行文库制备和细胞核测序之前，使用自动细胞计数器测量浓度并评估单细胞悬液的活力。生物信息学聚类揭示了与DG内已知细胞类型相对应的分离良好的细胞核组，有或没有FANS。在非FACS分选的样本中，大多数高质量的测序细胞核占神经元的84.9%。

它由三组神经元组成，表明齿状回中最具代表性的细胞群可能是颗粒神经元、其他兴奋性神经元和抑制性神经元。在非FACS分选的样品中，鉴定的非神经元簇主要由11.1%的神经胶质细胞类型组成，包括星形胶质细胞，少突胶质细胞和少突胶质细胞前体细胞，3.3%免疫细胞和0，6%Cajal-Retzius细胞。在进行FANS排除NeuN阳性群体的同时，神经胶质细胞簇变得突出，占总细胞核的81.3%。

对于每个细胞核 50， 000 个读数测序的样品，非 FACS 分选样品的每个细胞核检测到 25， 010 个基因，NeuN 阴性 FANS 样品检测到 1， 665.5 个基因，证实了单个细胞核的高质量转录组学分析和 FACS 分选不会破坏后续 snRNA-seq 的细胞核。非FACS分选样品中高比例的神经元具有更高的转录活性，每个细胞核2， 660个基因，非FACS分选样品中每个细胞核6， 170个转录本，平均转录活性为每个细胞核1， 090个基因，每个细胞核1， 785个转录本。一旦使用该协议生成数据，就值得考虑这些新的正交方法，例如特殊症状或体内研究以验证任何发现。

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