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
Summary
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。
Abstract
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。
Introduction
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。
Protocol
Representative Results
Discussion
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…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| 5 M Betaine | Sigma-Aldrich | Cat# B0300-5VL | |
| 10 mM dNTP mix | Thermo Fisher Scientific | Cat# R0192 | |
| 0.5 M EDTA, pH 8.0 | Thermo Fisher Scientific | Cat# 15575020 | |
| 10X Hanks’ Balanced Salt Solution | Thermo Fisher Scientific | Cat# 14185-052 | |
| 1 M HEPES | Thermo Fisher Scientific | Cat# 15630080 | |
| 1X KAPA HIFI Hotstart Master Mix | Kapa Biosciences | Cat# KK2602 | |
| 5 mL Round Bottom Polystyrene Tube, with Cell Strainer Cap | Corning | Cat# 352235 | |
| AATI, High Sensitivity NGS Fragment Analysis Kit (1 bp – 6,000 bp) | Advanced Analytical | Cat# DNF-474-1000 | |
| Bovine Serum Albumin | Sigma Aldrich | Cat# A8806 | |
| DNase I | Worthington | Cat# LS002007 | Working solution: 12500 units/ml |
| DTT, Molecular Grade | Promega | Cat# P1171 | |
| ERCC RNA Spike-In Mix | Thermo Fisher Scientific | Cat# 4456740 | |
| Fetal Bovine Serum | Thermo Fisher Scientific | Cat# 10437-028 | |
| Illumina XT Index Kit v2 Set A (96 indexes) | Illumina | Cat# FC-131-2001 | |
| Illumina XT Index Kit v2 Set B (96 indexes) | Illumina | Cat# FC-131-2002 | |
| Illumina XT Index Kit v2 Set C (96 indexes) | Illumina | Cat# FC-131-2003 | |
| Illumina XT Index Kit v2 Set D (96 indexes) | Illumina | Cat# FC-131-2004 | |
| Lambda Exonuclease (5 U/μl) | New England BioLabs | Cat# M0262S | |
| Mouse Fc block | BD Pharmingen | Cat# 553142 | |
| Myelin removal beads | Miltenyl Biotec | Cat# 130-096-433 | |
| Nextera XT DNA Sample Prep Kit | Illumina | Cat# FC-131-1096 | |
| NextSeq 500/550 High Output Kit v2.5 (150 Cycles) | Illumina | Cat# 20024907 | |
| PBS (10X), pH 7.4 | Thermo Fisher Scientific | Cat# 70011044 | |
| PCRClean DX beads | Aline Biosciences | Cat# C-1003-50 | |
| Propidium Iodide | Thermo Fisher Scientific | Cat# P3566 | Staining: 1:1000 |
| Qubit dsDNA HS Assay Kit | Thermal Fisher Scientific | Cat# Q32851 | |
| Rat monoclonal anti mouse/human CD11b, Brilliant Violet 421 (clone M1/70) | BioLegend | Cat# 101236; RRID: AB_11203704 | Staining: 1:300 |
| Rat monoclonal anti mouse CD45, PE/Cy7 (clone 30-F11) | Thermo Fisher Scientific | Cat# 25-0451-82; RRID: AB_469625 | Staining: 1:300 |
| Recombinant RNase Inhibitor | Takara Bio | Cat# 2313B | |
| SMARTScribe Reverse Transcriptase (100 U/μl) | Clontech | Cat# 639538 | Containing 5x First strand buffer |
| Oligonucleotides | |||
| 0.1 μM ISPCR Oligo: 5' – AAGCAGTGGTATCAA CGCAGAGT-3' |
(Picelli et al., 2014) | ||
| Oligo-dT30VN primer: 5' – AAGCAGTGGTATCAACGCA GAGTACT 30 VN-3' |
(Picelli et al., 2014) | ||
| TSO 5' – AAGCAGTGGTATCAACGCAGA GTACATrGrG+G-3' ("r" is forribobases and "+" is for an LNA base) |
(Picelli et al., 2014) | ||
| Solutions | |||
| FACS buffer | Recipe: sterile-filtered 1% FBS, 2 mM EDTA, 25 mM HEPES in 1X PBS | ||
| MCS buffer | Recipe: sterile-filtered 0.5% BSA, 2 mM EDTA in 1X PBS | ||
| Medium A | Recipe: 15 mM HEPES, 0.5% glucose in 1X HBSS without phenol red | ||
| Plates | |||
| 384-well Rigi-Plate PCR Microplates, Axygen Scientific | VWR | 89005-556 | |
| Hard-shell 96-well PCR plates | Bio-Rad | HSP9631 | |
| Others | |||
| Dumont #55 forceps | Fine Science Tools | 11295-51 | |
| Dounce homogenizer, 2 ml | Wheaton | 357422 | |
| Large depletion column | Miltenyi Biotec | 130-042-901 | |
| Large selection column | Miltenyi Biotec | 130-042-401 | |
| MACS MultiStand | Miltenyi Biotec | 130-042-303 | |
| QuadroMACS Separator | Miltenyi Biotec | 130-090-976 | |
| RNAzap | Thermo Fisher Scientific | AM9780 | |
| Strainer (70 μm) | Falcon | 352350 | |
| Equipment | |||
| BD FACSAria II | BD Biosciences | http://www.bdbiosciences.com/ | |
| Bioanalyzer | Agilent | 2100 | |
| Fragment Analyzer | Agilent | 5300 | |
| Mosquito HTS nanoliter pipetting robot | TTP Labtech | https://www.ttplabtech.com/ | |
| Qubit 4 Fluorometer | Thermo Fisher Scientific | Q33226 | |
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