人类胰腺胰岛的单细胞RNA测序与分析
Summary
在这里,我们提出了一个协议,利用基于液滴的微流体单细胞RNA测序技术,从分离的人类胰腺胰岛生成单个细胞的高质量、大规模转录组数据。
Abstract
胰腺胰岛由具有独特激素表达模式的内分泌细胞组成。内分泌细胞在适应正常和病理状况时表现出功能差异。该协议的目标是利用基于液滴的微流体单细胞RNA测序技术,生成每种内分泌细胞类型的高质量、大规模转录组数据。这些数据可用于在正常或特定条件下建立每种内分泌细胞类型的基因表达特征。该过程需要仔细操作、精确测量和严格的质量控制。在此协议中,我们描述了人类胰腺胰岛分离、测序和数据分析的详细步骤。约20,000个人类单小岛细胞的代表性结果表明,该协议的成功应用。
Introduction
胰腺胰岛释放内分泌激素来调节血糖水平。五种内分泌细胞类型在功能上和形态上各不相同,它们参与于这一基本作用:β细胞产生胰高血糖素、β细胞胰岛素、β细胞生长激素、PP细胞胰腺多肽和β-细胞ghrelin1。基因表达分析是在正常或特定条件下对内分泌细胞进行表征的有用方法。从历史上看,整个小岛基因表达分析是使用微阵列和下一代RNA测序2,3,4,5,6,7生成,8.虽然整个小岛转录组有助于识别器官特异性转录本和疾病候选基因,但它未能揭示每个小岛细胞类型的分子异质性。激光捕获微分型(LCM)技术已应用于直接从胰岛9、10、11、12获得特定细胞类型,但低于目标细胞的纯度人口。为了克服这些限制,荧光活性细胞分类(FACS)被用来选择特定的内分泌细胞群,如β-和β细胞13,14,15,16,17,18.此外,Dorrell等人使用基于抗体的FACS分类方法将β细胞分为四个亚群19。FACS排序的小岛细胞也可以镀出用于单个细胞的RNA测序;然而,基于板的方法在可伸缩性20,21,22方面面临着挑战。
为了生成各内分泌细胞类型的高质量、大规模转录组数据,我们应用微流体技术应用于人类小岛细胞。微流体平台以高通量、高质量和可扩展的方式23、24、25、26、27从大量单个细胞生成转录组数据。除了揭示大量捕获的细胞类型的分子特性外,高度可扩展的微流体平台还能在提供足够的细胞时识别稀有细胞类型。因此,该平台应用于人类胰腺胰岛允许分析ghrelin分泌β细胞,这是一种罕见的内分泌细胞类型,由于其稀缺性28,其功能鲜为人知。近年来,我们和其他一些人发表了几项研究,报告使用该技术29、30、31、32,人类胰岛的大规模转录数据。 33.这些数据是公开的,有用的资源为小岛社区研究内分泌细胞异质性及其在疾病的影响。
在这里,我们描述了一种基于液滴的微流体单细胞RNA测序协议,它被用于生成大约20,000个人类小岛细胞的转录组数据,包括β-、β-、β–、PP、β细胞,以及较小比例的非内分泌细胞32.工作流从孤立的人类胰岛开始,并描述了胰岛细胞分离、单细胞捕获和数据分析的步骤。该协议要求使用新鲜分离的小岛,并可应用于人类和其他物种(如啮齿动物)的小岛。使用此工作流,可以在基线和其他条件下构建无偏和全面的小岛细胞图集。
Protocol
1. 人类小岛分离 从15-80岁之间的男女尸体器官捐献者中分离出人类胰岛,没有预先存在的疾病,除非研究目的需要具有特定人口特征的捐赠者胰岛。 隔离后,将分离的小岛保存在组织培养设施中2-3天。通常需要超过 1 天的时间才能看到小岛损伤。 将小岛放入瓶子中,将其完全浸入胰岛介质中。通宵发货后,就把它运到实验室。 从胰岛供应商处获取已发货的小岛的胰岛等效?…
Representative Results
单细胞RNA测序工作流程包括三个步骤:将完整的人类胰岛分离成单个细胞悬浮液,使用基于液滴的技术捕获单个细胞,以及分析RNA-seq数据(图1)。首先,获得人类胰岛在一夜之间孵育。完整的胰岛在显微镜下被检查(图2A)。分离的斜岛细胞的完整性已经使用RNA荧光原位杂交(RNA-FISH)得到验证。如图2B所示,分别使?…
Discussion
近年来开发的单细胞技术为描述细胞类型和研究人类胰腺胰岛分子异质性提供了新的平台。我们采用了基于液滴的微流体单细胞分离和数据分析方案来研究人类胰岛。我们的协议成功地从超过20,000个单人类小岛细胞中生成了RNA测序数据,在序列质量和批次效应方面变化相对较小。
特别是,在该协议中,两个步骤对于高质量结果至关重要。分离人类胰岛时,需要谨慎。重要的是不要?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
没有
Materials
| 30 µm Pre-Separation Filters | Miltenyi Biotec | 130-041-407 | Cell strainer |
| 8-chamber slides | Chemometec | 102673-680 | Dell counting assay slides |
| Bioanalyzer High Sensitivity DNA Kit | Agilent | 5067-4626 | for QC |
| Bovine Serum Albumin | Sigma-Aldrich | A9647 | Single cell media |
| Chromium Single Cell 3' Library & Gel Bead Kit v2, 16 rxns | 10X Genomics | 120237 | Single cell reagents |
| Chromium Single Cell A Chip Kit v2, 48 rx (6 chips) | 10X Genomics | 120236 | Microfluidic chips |
| CMRL-1066 | ThermoFisher | 11530-037 | Complete islet media |
| EB Buffer | Qiagen | 19086 | Elution buffer |
| Eppendorf twin-tec PCR plate, 96-well, blue, semi-skirted | VWR | 47744-112 | Emulsion plate |
| Fetal Bovine Serum | ThermoFisher | 16000-036 | Complete islet media |
| Human islets | Prodo Labs | HIR | Isolated human islets |
| L-Glutamine (200 mM) | ThermoFisher | 25030-081 | Complete islet media |
| Nextera DNA Library Preparation Kit (96 samples) | Illumina | FC-121-1031 | Library preparation reagents |
| NextSeq 500/550 High Output Kit v2.5 (75 cycles) | Illumina | FC-404-2005 | Sequencing |
| Penicillin-Streptomycin (10,000 U/mL) | ThermoFisher | 15140-122 | Complete islet media |
| Qubit High Sensitivity dsDNA Kit | Life Technologies | Q32854 | for QC |
| Solution 18 | Chemometec | 103011-420 | Cell counting assay reagent |
| SPRISelect Reagent | Fisher Scientific | B23318 | Purification beads |
| Tissue Culture Dishes (10 cm) | VWR | 10861-594 | for islet culture |
| TrypLE Express | Life Technologies | 12604-013 | Cell dissociation solution |
| Zymo DNA Clean & Concentrator-5, 50 reactions | VWR | 77001-152 | Library clean up columns |
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Cite This Article
Xin, Y., Adler, C., Kim, J., Ding, Y., Ni, M., Wei, Y., Macdonald, L., Okamoto, H. Single-cell RNA Sequencing and Analysis of Human Pancreatic Islets. J. Vis. Exp. (149), e59866, doi:10.3791/59866 (2019).