Name: isolation of region-specific microglia from one adult mouse brain hemisphere for deep single-cell rna sequencing
Uploaded: 2019-12-03T13:00:00
Description: Watch this Scientific Journal Video about Isolation of Region-specific Microglia from One Adult Mouse Brain Hemisphere for Deep Single-cell RNA Sequencing at JoVE.com

我们感谢玛丽科·贝内特、利亚娜·妮可·博南诺和斯皮罗斯·达尔马尼斯在制定该协议期间提供的帮助。我们还感谢斯坦福共享FACS设施，特别是梅雷迪斯·韦格拉兹和丽莎·尼科尔斯;来自斯坦福蛋白质和核酸设施（PAN）的Yen Tran，迈克尔·埃卡特，因为他们对拍摄的大力支持。这项工作由JPB基金会和文森特·科茨基金会资助。

涉及啮齿动物的所有程序都符合斯坦福大学的指导方针，该准则符合国家和州法律和政策。所有动物程序都通过了斯坦福大学实验室动物护理行政小组的批准。
注：所有溶液和缓冲组合物均在材料表中提供。
1. 细胞隔离日的准备工作
<ol><li>准备以下试剂并将其冷却在冰上：中等A（50 mL）、磁活细胞分拣（MCS）缓冲液（30 mL）、F...

<ol>
	<li>Li, Q., Barres, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglia+and+macrophages+in+brain+homeostasis+and+disease.">Microglia and macrophages in brain homeostasis and disease.</a> Nature Reviews Immunology. 18 (4), 225-242 (2018).</li><li>Li, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+Heterogeneity+of+Microglia+and+Brain+Myeloid+Cells+Revealed+by+Deep+Single-Cell+RNA+Sequencing.">Developmental Heterogeneity of Microglia and Brain Myeloid Cells Revealed by Deep Single-Cell RNA Sequencing.</a> Neuron. 101 (2), 207-223 (2019).</li><li>Keren-Shaul, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Unique+Microglia+Type+Associated+with+Restricting+Development+of+Alzheimer's+Disease.">A Unique Microglia Type Associated with Restricting Development of Alzheimer's Disease.</a> Cell. 169 (7), 1276-1290 (2017).</li><li>Bohlen, C. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diverse+Requirements+for+Microglial+Survival,+Specification,+and+Function+Revealed+by+Defined-Medium+Cultures.">Diverse Requirements for Microglial Survival, Specification, and Function Revealed by Defined-Medium Cultures.</a> Neuron. 94 (4), 759-773 (2017).</li><li>Guerreiro, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TREM2+variants+in+Alzheimer's+disease.">TREM2 variants in Alzheimer's disease.</a> New England Journal of Medicine. 368 (2), 117-127 (2013).</li><li>Jonsson, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variant+of+TREM2+associated+with+the+risk+of+Alzheimer's+disease.">Variant of TREM2 associated with the risk of Alzheimer's disease.</a> New England Journal of Medicine. 368 (2), 107-116 (2013).</li><li>Zhang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+RNA-sequencing+transcriptome+and+splicing+database+of+glia,+neurons,+and+vascular+cells+of+the+cerebral+cortex.">An RNA-sequencing transcriptome and splicing database of glia, neurons, and vascular cells of the cerebral cortex.</a> Journal of Neuroscience. 34 (36), 11929-11947 (2014).</li><li>Colonna, M., Butovsky, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglia+Function+in+the+Central+Nervous+System+During+Health+and+Neurodegeneration.">Microglia Function in the Central Nervous System During Health and Neurodegeneration.</a> Annual Review of Immunology. 35, 441-468 (2017).</li><li>Bennett, M. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+tools+for+studying+microglia+in+the+mouse+and+human+CNS.">New tools for studying microglia in the mouse and human CNS.</a> Proceedings of the National Academy of Sciences of the United States of America. 113 (12), 1738-1746 (2016).</li><li>Hammond, T. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-Cell+RNA+Sequencing+of+Microglia+throughout+the+Mouse+Lifespan+and+in+the+Injured+Brain+Reveals+Complex+Cell-State+Changes.">Single-Cell RNA Sequencing of Microglia throughout the Mouse Lifespan and in the Injured Brain Reveals Complex Cell-State Changes.</a> Immunity. 50 (1), 253-271 (2019).</li><li>Van Hove, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+single-cell+atlas+of+mouse+brain+macrophages+reveals+unique+transcriptional+identities+shaped+by+ontogeny+and+tissue+environment.">A single-cell atlas of mouse brain macrophages reveals unique transcriptional identities shaped by ontogeny and tissue environment.</a> Nature Neuroscience. 22 (6), 1021-1035 (2019).</li><li>Chen, G., Ning, B., Shi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-Cell+RNA-Seq+Technologies+and+Related+Computational+Data+Analysis.">Single-Cell RNA-Seq Technologies and Related Computational Data Analysis.</a> Frontiers in Genetics. 10, 317 (2019).</li><li>Svensson, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Power+analysis+of+single-cell+RNA-sequencing+experiments.">Power analysis of single-cell RNA-sequencing experiments.</a> Nature Methods. 14 (4), 381-387 (2017).</li><li>Picelli, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Full-length+RNA-seq+from+single+cells+using+Smart-seq2.">Full-length RNA-seq from single cells using Smart-seq2.</a> Nature Protocols. 9 (1), 171-181 (2014).</li><li>Macosko, E. Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+Parallel+Genome-wide+Expression+Profiling+of+Individual+Cells+Using+Nanoliter+Droplets.">Highly Parallel Genome-wide Expression Profiling of Individual Cells Using Nanoliter Droplets.</a> Cell. 161 (5), 1202-1214 (2015).</li><li>Ziegenhain, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+Analysis+of+Single-Cell+RNA+Sequencing+Methods.">Comparative Analysis of Single-Cell RNA Sequencing Methods.</a> Molecular Cell. 65 (4), 631-643 (2017).</li><li>Tabula Muris, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+transcriptomics+of+20+mouse+organs+creates+a+Tabula+Muris.">Single-cell transcriptomics of 20 mouse organs creates a Tabula Muris.</a> Nature. 562 (7727), 367-372 (2018).</li><li>Bohlen, C. J., Bennett, F. C., Bennett, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+Culture+of+Microglia.">Isolation and Culture of Microglia.</a> Current Protocols in Immunology. 125 (1), 70 (2018).</li><li>Haimon, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Re-evaluating+microglia+expression+profiles+using+RiboTag+and+cell+isolation+strategies.">Re-evaluating microglia expression profiles using RiboTag and cell isolation strategies.</a> Nature Immunology. 19 (6), 636-644 (2018).</li><li>Collins, H. Y., Bohlen, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+Culture+of+Rodent+Microglia+to+Promote+a+Dynamic+Ramified+Morphology+in+Serum-free+Medium.">Isolation and Culture of Rodent Microglia to Promote a Dynamic Ramified Morphology in Serum-free Medium.</a> Journal of Visualized Experiments. (133), e57122 (2018).</li><li>Nikodemova, M., Watters, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+isolation+of+live+microglia+with+preserved+phenotypes+from+adult+mouse+brain.">Efficient isolation of live microglia with preserved phenotypes from adult mouse brain.</a> Journal of Neuroinflammation. 9, 147 (2012).</li><li>Swartzlander, D. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concurrent+cell+type-specific+isolation+and+profiling+of+mouse+brains+in+inflammation+and+Alzheimer's+disease.">Concurrent cell type-specific isolation and profiling of mouse brains in inflammation and Alzheimer's disease.</a> Journal of Clinical Investigation Insight. 3 (13), 121109 (2018).</li><li>Hennig, B. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-Scale+Low-Cost+NGS+Library+Preparation+Using+a+Robust+Tn5+Purification+and+Tagmentation+Protocol.">Large-Scale Low-Cost NGS Library Preparation Using a Robust Tn5 Purification and Tagmentation Protocol.</a> G3. 8 (1), 79-89 (2018).</li></ol>

微胶质积极与中枢神经系统中的其他细胞类型相互作用，并且对环境刺激非常敏感。为了尽量减少在分离过程中其基因表达的炎症反应和异常变化，该协议从先前公布的方法9进行了简化，现在适合从单个小鼠大脑半球的多个区域并行分离微胶质。组织和试剂保存在低温下，实验及时进行（从解剖到分拣约3.5小时），根据有限的组织尺寸，处理次数更少，试剂较少。我们选择其他...

微胶质，占所有神经细胞的5%-10%，是分散在中枢神经系统（CNS）1的常驻巨噬细胞。在血脑屏障的背后，健康成人大脑中典型的微胶质包含许多精细过程，这些过程会迅速延伸和缩回，与脑血管瘤中的神经元和其他胶质细胞相互作用。微胶质也可以采用与在特定发育阶段或损伤和疾病1、2、3、4中的免疫挑战相关的与增加的咽喉功能相关的阿米巴细胞形态。最近令人振奋的发现清楚地表明，微胶质绝不是大脑衍生或病理信号的被动旁观者，但在控制大脑发育和平衡方面起着关键作用，例如，通过支持神经元生存、修剪未成熟的突触、促进寡核苷酸系细胞分化以及血管生成1。随着微胶质的更多功能被阐明，兴奋进一步由人类遗传学研究，这表明，许多神经退行性疾病风险基因，如TREM2，主要或完全表达微胶质5，6，7。鉴于它们在发育中的重要性和合理的疾病驱动作用，我们最近为理解微胶质基因调控和功能而付出巨大努力，以期找到神经退行性疾病1、8的新治疗靶点。
RNA测序（RNA-seq）允许对细胞类型特异性基因表达进行无偏的表征，这反过来又指导科学家研究致密细胞网络中的基因功能7。RNA-seq主要在大体积样品上进行，导致发现一种静温微胶质基因特征，使其与其他神经和免疫细胞9区分开来。然而，这种方法可以忽视微胶质之间的分子和功能差异，特别是那些暂时存在于发育中，或与衰老和疾病相关的差异。事实上，单细胞RNA-seq（scRNA-seq）提供了敏感性和分辨率，通过揭示以前未被认识的微胶质异质性，在各种上下文中2，3，10，彻底改变了该领域。此外，由于在CNS循环界面中存在其他类似的免疫细胞，scRNA-seq提供了信息，有助于设计新的工具，以分离和功能解剖这些相关细胞，而事先知之甚少2，11。
已经发明了一系列不同的scRNA-seq平台，每个平台都适合某些应用12。通常，基于滴滴的方法（如 10x 基因组学）的吞吐量较高，每次运行中测序的 （数千个）细胞，并且对于可能包含需要广泛分类的混合细胞群的输入的选择性较低。基于板的方法提供更高的灵敏度和读取深度13，14，通常针对特定人群从细胞排序，以揭示细微的差异或罕见的成绩单。鉴于在所有CNS细胞类型中微胶质细胞，特别是发育或疾病相关亚群的较小比例，通常可取地将微胶质从特定感兴趣的区域分离，并获取深度和全长转录组信息，以了解其异质性。
在这里，我们详细介绍了如何从从单个半球解剖的不同小鼠大脑区域分离微胶质，这些区域在半自动基于板的库制备程序后用于单细胞（或散装）RNA-seq。另一半球则可用于组织学验证。这种分离方案从先前公布的方法9中简化，旨在最大化少量起始材料的产量，同时维持内源性微胶质基因表达谱。我们使用荧光活性细胞分拣（FACS）将微胶质（或其他相关的相关免疫细胞）浓缩到96孔板中，并将试剂体积小型化，用于库制备，以提高产量。我们重点介绍这个敏感的scRNA-seq平台，尽管可以应用其他基于板的策略。这种方法可以很容易地适应，以分离微胶质从其他解剖组织，如损伤或疾病病灶，和小鼠的年龄可以改变几乎任何产后阶段。单细胞转录组学研究对区域微胶质的有效分离将有助于更好地了解其在健康和疾病中的功能。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>5 M Betaine</td>
			<td>Sigma-Aldrich</td>
			<td>Cat# B0300-5VL</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mM dNTP mix</td>
			<td>Thermo Fisher Scientific</td>
			<td>Cat# R0192</td>
			<td></td>
		</tr>
		<tr>
			<td>0.5 M EDTA, pH 8.0</td>
			<td>Thermo Fisher Scientific</td>
			<td>Cat# 15575020</td>
			<td></td>
		</tr>
		<tr>
			<td>10X Hanks' Balanced Salt Solution</td>
			<td>Thermo Fisher Scientific</td>
			<td>Cat# 14185-052</td>
			<td></td>
		</tr>
		<tr>
			<td>1 M HEPES</td>
			<td>Thermo Fisher Scientific</td>
			<td>Cat# 15630080</td>
			<td></td>
		</tr>
		<tr>
			<td>1X KAPA HIFI Hotstart Master Mix</td>
			<td>Kapa Biosciences</td>
			<td>Cat# KK2602</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL Round Bottom Polystyrene Tube, with Cell Strainer Cap</td>
			<td>Corning</td>
			<td>Cat# 352235</td>
			<td></td>
		</tr>
		<tr>
			<td>AATI, High Sensitivity NGS Fragment Analysis Kit (1 bp &#8211; 6,000 bp)</td>
			<td>Advanced Analytical</td>
			<td>Cat# DNF-474-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma Aldrich</td>
			<td>Cat# A8806</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase I</td>
			<td>Worthington</td>
			<td>Cat# LS002007</td>
			<td>Working solution: 12500 units/ml</td>
		</tr>
		<tr>
			<td>DTT, Molecular Grade</td>
			<td>Promega</td>
			<td>Cat# P1171</td>
			<td></td>
		</tr>
		<tr>
			<td>ERCC RNA Spike-In Mix</td>
			<td>Thermo Fisher Scientific</td>
			<td>Cat# 4456740</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Thermo Fisher Scientific</td>
			<td>Cat# 10437-028</td>
			<td></td>
		</tr>
		<tr>
			<td>Illumina XT Index Kit v2 Set A (96 indexes)</td>
			<td>Illumina</td>
			<td>Cat# FC-131-2001</td>
			<td></td>
		</tr>
		<tr>
			<td>Illumina XT Index Kit v2 Set B (96 indexes)</td>
			<td>Illumina</td>
			<td>Cat# FC-131-2002</td>
			<td></td>
		</tr>
		<tr>
			<td>Illumina XT Index Kit v2 Set C (96 indexes)</td>
			<td>Illumina</td>
			<td>Cat# FC-131-2003</td>
			<td></td>
		</tr>
		<tr>
			<td>Illumina XT Index Kit v2 Set D (96 indexes)</td>
			<td>Illumina</td>
			<td>Cat# FC-131-2004</td>
			<td></td>
		</tr>
		<tr>
			<td>Lambda Exonuclease (5 U/&#956;l)</td>
			<td>New England BioLabs</td>
			<td>Cat# M0262S</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse Fc block</td>
			<td>BD Pharmingen</td>
			<td>Cat# 553142</td>
			<td></td>
		</tr>
		<tr>
			<td>Myelin removal beads</td>
			<td>Miltenyl Biotec</td>
			<td>Cat# 130-096-433</td>
			<td></td>
		</tr>
		<tr>
			<td>Nextera XT DNA Sample Prep Kit</td>
			<td>Illumina</td>
			<td>Cat# FC-131-1096</td>
			<td></td>
		</tr>
		<tr>
			<td>NextSeq 500/550 High Output Kit v2.5 (150 Cycles)</td>
			<td>Illumina</td>
			<td>Cat# 20024907</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS (10X), pH 7.4</td>
			<td>Thermo Fisher Scientific</td>
			<td>Cat# 70011044</td>
			<td></td>
		</tr>
		<tr>
			<td>PCRClean DX beads</td>
			<td>Aline Biosciences</td>
			<td>Cat# C-1003-50</td>
			<td></td>
		</tr>
		<tr>
			<td>Propidium Iodide</td>
			<td>Thermo Fisher Scientific</td>
			<td>Cat# P3566</td>
			<td>Staining: 1:1000</td>
		</tr>
		<tr>
			<td>Qubit dsDNA HS Assay Kit</td>
			<td>Thermal Fisher Scientific</td>
			<td>Cat# Q32851</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat monoclonal anti mouse/human CD11b, Brilliant Violet 421 (clone M1/70)</td>
			<td>BioLegend</td>
			<td>Cat# 101236; RRID: AB_11203704</td>
			<td>Staining: 1:300</td>
		</tr>
		<tr>
			<td>Rat monoclonal anti mouse CD45, PE/Cy7 (clone 30-F11)</td>
			<td>Thermo Fisher Scientific</td>
			<td>Cat# 25-0451-82; RRID: AB_469625</td>
			<td>Staining: 1:300</td>
		</tr>
		<tr>
			<td>Recombinant RNase Inhibitor</td>
			<td>Takara Bio</td>
			<td>Cat# 2313B</td>
			<td></td>
		</tr>
		<tr>
			<td>SMARTScribe Reverse Transcriptase (100 U/&#956;l)</td>
			<td>Clontech</td>
			<td>Cat# 639538</td>
			<td>Containing 5x First strand buffer</td>
		</tr>
		<tr>
			<td colspan="4">Oligonucleotides</td>
		</tr>
		<tr>
			<td>0.1 &#956;M ISPCR Oligo: 5&#39; - AAGCAGTGGTATCAA CGCAGAGT-3&#39;</td>
			<td></td>
			<td></td>
			<td>(Picelli et al., 2014)</td>
		</tr>
		<tr>
			<td>Oligo-dT30VN primer: 5&#39; - AAGCAGTGGTATCAACGCA GAGTACT 30 VN-3&#39;</td>
			<td></td>
			<td></td>
			<td>(Picelli et al., 2014)</td>
		</tr>
		<tr>
			<td>TSO 5&#39; - AAGCAGTGGTATCAACGCAGA GTACATrGrG+G-3&#39; (&#34;r&#34; is forribobases and &#34;+&#34; is for an LNA base)</td>
			<td></td>
			<td></td>
			<td>(Picelli et al., 2014)</td>
		</tr>
		<tr>
			<td colspan="4">Solutions</td>
		</tr>
		<tr>
			<td>FACS buffer</td>
			<td></td>
			<td></td>
			<td>Recipe: sterile-filtered 1% FBS, 2 mM EDTA, 25 mM HEPES in 1X PBS</td>
		</tr>
		<tr>
			<td>MCS buffer</td>
			<td></td>
			<td></td>
			<td>Recipe: sterile-filtered 0.5% BSA, 2 mM EDTA in 1X PBS</td>
		</tr>
		<tr>
			<td>Medium A</td>
			<td></td>
			<td></td>
			<td>Recipe: 15 mM HEPES, 0.5% glucose in 1X HBSS without phenol red</td>
		</tr>
		<tr>
			<td colspan="4">Plates</td>
		</tr>
		<tr>
			<td>384-well Rigi-Plate PCR Microplates, Axygen Scientific</td>
			<td>VWR</td>
			<td>89005-556</td>
			<td></td>
		</tr>
		<tr>
			<td>Hard-shell 96-well PCR plates</td>
			<td>Bio-Rad</td>
			<td>HSP9631</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">Others</td>
		</tr>
		<tr>
			<td>Dumont #55 forceps</td>
			<td>Fine Science Tools</td>
			<td>11295-51</td>
			<td></td>
		</tr>
		<tr>
			<td>Dounce homogenizer, 2 ml</td>
			<td>Wheaton</td>
			<td>357422</td>
			<td></td>
		</tr>
		<tr>
			<td>Large depletion column</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-901</td>
			<td></td>
		</tr>
		<tr>
			<td>Large selection column</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-401</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS MultiStand</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-303</td>
			<td></td>
		</tr>
		<tr>
			<td>QuadroMACS Separator</td>
			<td>Miltenyi Biotec</td>
			<td>130-090-976</td>
			<td></td>
		</tr>
		<tr>
			<td>RNAzap</td>
			<td>Thermo Fisher Scientific</td>
			<td>AM9780</td>
			<td></td>
		</tr>
		<tr>
			<td>Strainer (70 &#956;m)</td>
			<td>Falcon</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">Equipment</td>
		</tr>
		<tr>
			<td>BD FACSAria II</td>
			<td>BD Biosciences</td>
			<td><a href="http://www.bdbiosciences.com/documents/BD_FACSAria_II_cell_sorter_brochure.pdf" target="_blank">http://www.bdbiosciences.com/</a></td>
			<td></td>
		</tr>
		<tr>
			<td>Bioanalyzer</td>
			<td>Agilent</td>
			<td>2100</td>
			<td></td>
		</tr>
		<tr>
			<td>Fragment Analyzer</td>
			<td>Agilent</td>
			<td>5300</td>
			<td></td>
		</tr>
		<tr>
			<td>Mosquito HTS nanoliter pipetting robot</td>
			<td>TTP Labtech</td>
			<td><a href="https://www.ttplabtech.com/products/liquid-handling/mosquito-hts/" target="_blank">https://www.ttplabtech.com/</a></td>
			<td></td>
		</tr>
		<tr>
			<td>Qubit 4 Fluorometer</td>
			<td>Thermo Fisher Scientific</td>
			<td>Q33226</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of region-specific microglia from one adult mouse brain hemisphere for deep single-cell rna sequencing

作为中枢神经系统中的常住巨噬细胞，微胶质能积极控制大脑发育和平衡，其功能障碍可能导致人类疾病。在发现静温微胶质的分子特征以及其基因表达的变化以响应环境刺激方面取得了相当大的进展。随着单细胞基因组方法的出现和成熟，人们越来越认识到，异质微胶质可能是它们在不同发育和病理条件下所发挥的不同作用的基础。通过有效分离微胶质与特定感兴趣区域，然后对单个细胞进行敏感分析，可以进一步剖析这种异质性。在这里，我们提供了一个详细的协议，从单个成年小鼠大脑半球的不同大脑区域快速分离微胶质。我们还演示了如何使用这些排序的微胶用于基于板的深层单细胞RNA测序。我们讨论了此方法对其他场景的适应性，并为改进系统以适应大规模研究提供了指南。

该协议描述了一种在一个成人注入脑半球中从不同大脑区域分离和排序微胶质的方法，然后是scRNA-seq。我们使用点心来创建单个细胞悬浮液，并作为丰富微胶质的第一步。不足或过度采用会降低产量。此外，成年小鼠大脑含有高水平的骨髓，如果去除不当，也会降低分拣效率和产量。因此，在进行抗体染色之前，我们使用锥蓝色锥形和血细胞计来估计骨髓去除的产量、细胞活力和疗效（步骤4.9）?...

Watch this Scientific Journal Video about Isolation of Region-specific Microglia from One Adult Mouse Brain Hemisphere for Deep Single-cell RNA Sequencing at JoVE.com

Isolation of Region-specific Microglia from One Adult Mouse Brain Hemisphere for Deep Single-cell RNA Sequencing

我们为从成年小鼠大脑半球的不同解剖区域分离微胶质提供方案，然后为全长转录体进行深度单细胞RNA测序的半自动库制备。该方法将有助于阐明微胶质在健康和疾病中的功能异质性。

从一个成年小鼠脑半球分离区域特异性微胶质，进行深度单细胞RNA测序

As resident macrophages in the central nervous system, microglia actively control brain development and homeostasis, and their dysfunctions may drive ...

这种方法允许在单细胞水平上无偏地捕获微胶质基因表达，这可以帮助阐明健康而患病大脑中微胶质的分子异质性。该技术的主要优点是，它提供了从不同大脑区域快速分离微胶质的详细程序，并展示了如何有效地生成用于深度测序的单细胞RA6库的播放基础。要开始此过程，请准备文本协议中概述的所有需要的缓冲区和解决方案，并在冰上冷却它们。安乐死和斩首鼠标后，用小剪刀切开头部的皮肤，露出下面的头骨。然后，切穿下垂缝合线、羊皮缝合线和冠状缝合线。使用钳子在不损害组织的情况下拉出双骨和异形骨的两侧，并小心地将大脑移入含有中等 A 的解剖培养皿中。使用数字 55 钳子将小脑与皮质叶和脑干分离。然后，使用数字55钳仔细解剖出海马和线状体从皮层，并转移每个组织到一个单独的集合培养皿。首先，使用剃刀刀片将每个大脑区域切成小于一立方毫米的细片。使用带尖端的一毫升移液器，将组织件转移到预冷却的 Dounce 均质器中。通过缓慢地将活塞旋转到 Dounce 均质器中，使组织均匀，进行 6 到 10 次全冲程，直到不存在可见的块。然后，通过70微米过滤器将分离的组织转移到50毫升管中。用总共六毫升的冷介质 A 冲洗每个 Dounce 均质器和活塞，然后将冲洗转移到 15 毫升管中进行离心。在400倍g和4摄氏度下将单细胞悬浮液离心5分钟，5分钟休息。在磁性分离器中用三毫升 MCS 冲洗一个大损耗柱和三个大选择柱。当组织样品的离心完成时，移液器取出并丢弃上流液，而不会干扰颗粒。在含有RNase抑制剂的MCS缓冲液中重新填充细胞。将100微升的米林去除珠添加到每个管中，每个管中含有从皮层和小脑中再悬浮的细胞，并在每个管中加入50微升的米林去除珠，每个管中含有从海马和色相体中再悬浮的细胞。在冰上孵化管子10分钟。在此之后，将MCS添加到含有皮质细胞的管子中，使其体积达到两毫升，并加入剩余的管子，使每个体积达到一毫升。一旦柱为空的冲洗缓冲液，将两毫升皮质细胞加载到大损耗柱上，将彼此一毫升的细胞悬浮加载到单独的大选择柱上。接下来，用一毫升的 MCS 缓冲液清洗大耗竭柱，每次洗涤时使用一毫升 MCS 缓冲液洗涤每个大选择柱两次。通过 35 微升滤机盖过滤细胞到圆底 FACS 管中。这些管子在400倍g和4摄氏度下离心5分钟，以产生细胞。然后，慢慢倒出上根，在纸巾上涂抹管子的边缘。用 300 微升的 FACS 缓冲液重新填充每个管中的细胞。首先，在每个管中加入5微升小鼠FC受体块试剂。在冰上孵育五分钟。然后，在每个管中添加一个 CD45 PE 尺寸 7 微升和 CD11B BV421 微升。在室温下将管子孵育在摇床上10分钟。在此之后，添加两毫升的 FACS 缓冲液进行洗涤。在400倍g和4摄氏度下离心5分钟，以产生细胞的颗粒。用含有一微升的RNase抑制剂和0.5微升的碘化丙胺的FACS缓冲液，在每管中重新填充细胞。按照标准的 FACS 程序，将带 100 微米喷嘴的单活微胶质分拣到 96 孔 PCR 板中，每个孔中包含四个微升的解液缓冲液。简要地旋转板，并使用台顶离心机旋转它们。将盘子存放在零下80摄氏度，直到库准备。准备好继续操作时，在冰上解冻盘子。使用热循环器执行第一个转录，以生成cDNA，并放大cDNA与额外的外核素酶消化步骤，如文本协议中概述。之后，通过在每个井中加入18微升磁珠来净化cDNA。在磁性支架上孵育板五分钟。然后，取出上一液，用新鲜制作的80%乙醇洗两次样品，每次洗净时用80微升乙醇。要进行标记，请使用纳米光移液机将每个 cDNA 样品的 0.4 微升与 1.2 微升 Tn5 标记试剂混合在 384 井板中，在 55 摄氏度下进行 10 分钟。要停止标记反应，在室温下加入0.4微升中和缓冲液5分钟。接下来，在384个索引和1.2微升PCR混合到样本和放大库，如文本协议中概述。将同一 384 井板的所有独立库汇集在一起，并使用磁珠来净化最终的池库。在这项研究中，微胶质从成年小鼠大脑半球的皮层、小脑、海马和线状体中分离。在显微镜下，使用锥蓝和血细胞计对准备好的细胞悬浮液进行检查，以估计在进行抗体染色之前清除米林的产率、细胞活力和功效。此时，皮层的总细胞计数应超过30，000，其他组织应超过5，000。超过90%的细胞应该是可行的与小梅林碎片。FACS 机器用于对微胶质进行排序，微胶质通常是 CD45 低和 CD11B 正。成功分离应产生超过80%的微胶质出所有活的单细胞，至少对于皮质组织。一旦单个微胶质被捕获到解解缓冲液中，RNA被释放，然后反向转录到cDNA，然后被放大23个周期。作为毛细管电泳平台，碎片分析仪和高灵敏度NGS片段套件提供有关尺寸分布的快速、准确信息，以及96井板每个井中存在的cDNA分子数量。显示500到5000个碱基对之间的污迹样本，以及高于一定浓度阈值的样本可用于制作库。这些样本被测序到每个细胞超过一百万的原始读取深度，从而饱和了单细胞RNA测序方法的检测能力。每个微胶质细胞的映射率约为60%，超过2000个基因。以前从这种分离方法生成的数据通过独立实验复制，证明了在序列种群中检测微胶质特异性基因的敏感性。通过单微胶质转录的信息，研究人员可以在他们感兴趣的环境中发现独特的细胞群，并进一步研究这些新识别的细胞群的功能相关性。

Research

JoVE Journal

Neuroscience

Non-Laser Capture Microscopy Approach for the Microdissection of Discrete Mouse Brain Regions for Total RNA Isolation and Downstream Next-Generation Sequencing and Gene Expression Profiling

Non-Laser Capture Microscopy Approach for the Microdissection of Discrete Mouse Brain Regions for Total RNA Isolation and Downstream Next-Generation Sequencing and Gene Expression Profiling  |

总RNA的分离和下游下一代测序和基因表达谱的非激光捕获显微切割离散小鼠脑地区显微镜方法

As technological platforms, approaches such as next-generation sequencing, microarray, and qRT-PCR have great promise for expanding our understanding of ...

科研

神经科学

One Mouse, Two Cultures: Isolation and Culture of Adult Neural Stem Cells from the Two Neurogenic Zones of Individual Mice

一个鼠标，两种文化：隔离和成人神经的培养干细胞从单个小鼠的两个神经源性区

The neurosphere assay and the adherent monolayer culture system are valuable tools to determine the potential (proliferation or differentiation) of adult ...

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

大脑切片生物素化：一个<em&gt;前体内</em&gt;方法来衡量地区特有的质膜蛋白贩运成年神经元

Regulated endocytic trafficking is the central mechanism facilitating a variety of neuromodulatory events, by dynamically controlling receptor, ion ...

Characterization and Isolation of Mouse Primary Microglia by Density Gradient Centrifugation

密度梯度离心法分离小鼠原发胶质细胞的研究

Microglia, the resident immune cells in the brain, are the first responders to inflammation or injury in the central nervous system. Recent research has ...

Isolation and RNA Extraction of Neurons, Macrophages and Microglia from Larval Zebrafish Brains

斑马鱼脑幼虫神经元、巨噬细胞和小胶质的分离和 RNA 提取

To gain a detailed understanding of the role of different CNS cells during development or the establishment and progression of brain pathologies, it is ...

Isolation of Adult Spinal Cord Nuclei for Massively Parallel Single-nucleus RNA Sequencing

Probing an individual cell&#39;s gene expression enables the identification of cell type and cell state. Single-cell RNA sequencing has emerged as a ...

Single-cell RNA Sequencing of Fluorescently Labeled Mouse Neurons Using Manual Sorting and Double In Vitro Transcription with Absolute Counts Sequencing (DIVA-Seq)

Single-cell RNA Sequencing of Fluorescently Labeled Mouse Neurons Using Manual Sorting and Double In Vitro Transcription with Absolute Counts Sequencing DIVA-Seq

荧光标记小鼠神经元的单细胞 RNA 序列的人工分选和双体外转录与绝对计数测序 (天后-Seq)

Single-cell RNA sequencing (RNA-seq) is now a widely implemented tool for assaying gene expression. Commercially available single-cell RNA-sequencing ...

Assessment of Long-term Depression Induction in Adult Cerebellar Slices

成人脑切片长期抑郁症诱导评估

Synaptic plasticity provides a mechanism for learning and memory. For cerebellar motor learning, long-term depression (LTD) of synaptic transmissions from ...