Name: single-cell transcriptomic analyses of mouse pancreatic endocrine cells
Uploaded: 2018-09-30T13:00:00
Description: Watch this Scientific Journal Video about Single-cell Transcriptomic Analyses of Mouse Pancreatic Endocrine Cells at JoVE.com

我们感谢国家蛋白质科学中心, 北京 (北京大学) 和北京-清华生命科学计算平台中心。这项工作得到中国科学技术部 (2015CB942800)、中国国家自然科学基金 (31521004、31471358和 31522036) 的支持, 并由北京-清华大学生命科学中心向 C. R.X. 提供资助。

这里所描述的所有方法都已获得北京大学机构动物护理和使用委员会 (IACUC) 的批准。
1. 胰腺隔离
<ol><li>
为 E17.5 (胚胎天 17.5) 胚胎:
	<ol>
<li>估计胚胎日0.5 根据时间点时, 阴道插头出现。</li>
		<li>通过2的管理, 牺牲怀孕的老鼠。用70% 酒精喷雾腹部毛皮。</li>
		<li>用剪刀从延伸到肋骨的生殖器区做一个 V 形切口。这个过程将完全打开腹腔。</li>
		<li>解剖子宫...

<ol>
	<li>Gu, G., Dubauskaite, J., Melton, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+evidence+for+the+pancreatic+lineage:+NGN3++cells+are+islet+progenitors+and+are+distinct+from+duct+progenitors.">Direct evidence for the pancreatic lineage: NGN3+ cells are islet progenitors and are distinct from duct progenitors.</a> Development. 129 (10), 2447-2457 (2002).</li><li>Oliver-Krasinski, J. M., Stoffers, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+origin+of+the+beta+cell.">On the origin of the beta cell.</a> Genes &amp; Development. 22 (15), 1998-2021 (1998).</li><li>Dor, Y., Brown, J., Martinez, O. I., Melton, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+pancreatic+beta-cells+are+formed+by+self-duplication+rather+than+stem-cell+differentiation.">Adult pancreatic beta-cells are formed by self-duplication rather than stem-cell differentiation.</a> Nature. 429 (6987), 41-46 (2004).</li><li>Smukler, S. 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=The+adult+mouse+and+human+pancreas+contain+rare+multipotent+stem+cells+that+express+insulin.">The adult mouse and human pancreas contain rare multipotent stem cells that express insulin.</a> Cell Stem Cell. 8 (3), 281-293 (2011).</li><li>Dorrell, 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=Human+islets+contain+four+distinct+subtypes+of+beta+cells.">Human islets contain four distinct subtypes of beta cells.</a> Nature Communications. 7, 11756 (2016).</li><li>Bader, E., 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=Identification+of+proliferative+and+mature+beta-cells+in+the+islets+of+Langerhans.">Identification of proliferative and mature beta-cells in the islets of Langerhans.</a> Nature. 535 (7612), 430-434 (2016).</li><li>Saliba, A. E., Westermann, A. J., Gorski, S. A., Vogel, J. <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:+Advances+and+future+challenges.">Single-cell RNA-seq: Advances and future challenges.</a> Nucleic Acids Research. 42 (14), 8845-8860 (2014).</li><li>Qiu, W. 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=Deciphering+pancreatic+islet+beta+cell+and+alpha+cell+maturation+pathways+and+characteristic+features+at+the+single-cell+level.">Deciphering pancreatic islet beta cell and alpha cell maturation pathways and characteristic features at the single-cell level.</a> Cell Metabolism. 25 (5), 1194-1205 (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=Smart-seq2+for+sensitive+full-length+transcriptome+profiling+in+single+cells.">Smart-seq2 for sensitive full-length transcriptome profiling in single cells.</a> Nature Methods. 10 (11), 1096-1098 (2013).</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>Piccand, 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=Pak3+promotes+cell+cycle+exit+and+differentiation+of+beta-cells+in+the+embryonic+pancreas+and+is+necessary+to+maintain+glucose+homeostasis+in+adult+mice.">Pak3 promotes cell cycle exit and differentiation of beta-cells in the embryonic pancreas and is necessary to maintain glucose homeostasis in adult mice.</a> Diabetes. 63 (1), 203-215 (2014).</li><li>Veite-Schmahl, M. J., Regan, D. P., Rivers, A. C., Nowatzke, J. F., Kennedy, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissection+of+the+mouse+pancreas+for+histological+analysis+and+metabolic+profiling.">Dissection of the mouse pancreas for histological analysis and metabolic profiling.</a> Journal of Visualized Experiments. (126), (2017).</li><li>Hu, P., Zhang, W., Xin, H., Deng, G. <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+isolation+and+analysis.">Single cell isolation and analysis.</a> Frontiers in Cell and Developmental Biology. 4, 116 (2016).</li><li>Haque, A., Engel, J., Teichmann, S. A., Lonnberg, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+practical+guide+to+single-cell+RNA-sequencing+for+biomedical+research+and+clinical+applications.">A practical guide to single-cell RNA-sequencing for biomedical research and clinical applications.</a> Genome Medicine. 9 (1), 75 (2017).</li><li>. FastQC: A quality control tool for high throughput sequence data Available from: <a target="_blank" href="https://www.bioinformatics.babraham.ac.uk/projects/fastqc/">https://www.bioinformatics.babraham.ac.uk/projects/fastqc/</a> (2010)</li><li>Langmead, B., Salzberg, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+gapped-read+alignment+with+Bowtie+2.">Fast gapped-read alignment with Bowtie 2.</a> Nature Methods. 9 (4), 357-359 (2012).</li><li>Kim, D., 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=TopHat2:+accurate+alignment+of+transcriptomes+in+the+presence+of+insertions,+deletions+and+gene+fusions.">TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions.</a> Genome Biology. 14 (4), (2013).</li><li>Anders, S., Pyl, P. T., Huber, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HTSeq--a+Python+framework+to+work+with+high-throughput+sequencing+data.">HTSeq--a Python framework to work with high-throughput sequencing data.</a> Bioinformatics. 31 (2), 166-169 (2015).</li><li>Wagner, G. P., Kin, K., Lynch, V. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+mRNA+abundance+using+RNA-seq+data:+RPKM+measure+is+inconsistent+among+samples.">Measurement of mRNA abundance using RNA-seq data: RPKM measure is inconsistent among samples.</a> Theory in Biosciences. 131 (4), 281-285 (2012).</li><li>Brennecke, 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=Accounting+for+technical+noise+in+single-cell+RNA-seq+experiments.">Accounting for technical noise in single-cell RNA-seq experiments.</a> Nature Methods. 10 (11), 1093-1095 (2013).</li><li>Le, S., Josse, J., Husson, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FactoMineR:+An+R+package+for+multivariate+analysis.">FactoMineR: An R package for multivariate analysis.</a> Journal of Statistical Software. 25 (1), (2008).</li><li>Hadley, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> ggplot2: Elegant graphics for data analysis. , (2009).</li><li>. gplots: Various R Programming Tools for Plotting Data Available from: <a target="_blank" href="https://cran.r-project.org/package=gplots">https://cran.r-project.org/package=gplots</a> (2016)</li><li>Marcheva, 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=Disruption+of+the+clock+components+CLOCK+and+BMAL1+leads+to+hypoinsulinaemia+and+diabetes.">Disruption of the clock components CLOCK and BMAL1 leads to hypoinsulinaemia and diabetes.</a> Nature. 466 (7306), 627-631 (2010).</li><li>Li, 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=Single-cell+RNA-seq+analysis+maps+development+of+human+germline+cells+and+gonadal+niche+interactions.">Single-cell RNA-seq analysis maps development of human germline cells and gonadal niche interactions.</a> Cell Stem Cell. , (2017).</li><li>Qi, M., 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=Human+pancreatic+islet+isolation:+Part+I:+Digestion+and+collection+of+pancreatic+tissue.">Human pancreatic islet isolation: Part I: Digestion and collection of pancreatic tissue.</a> Journal of Visualized Experiments. (27), (2009).</li><li>Qi, M., 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=Human+pancreatic+islet+isolation:+Part+II:+Purification+and+culture+of+human+islets.">Human pancreatic islet isolation: Part II: Purification and culture of human islets.</a> Journal of Visualized Experiments. (27), (2009).</li><li>Teo, A. K. K., 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+analyses+of+human+islet+cells+reveal+de-differentiation+signatures.">Single-cell analyses of human islet cells reveal de-differentiation signatures.</a> Cell Death Discovery. 4 (14), (2018).</li></ol>

在本协议中, 我们展示了一种有效且易于使用的方法来研究胰β细胞的单细胞表达谱。该方法可用于分离胚胎、新生儿和产后胰腺的内分泌细胞, 并进行单细胞 transcriptomic 分析。
最关键的步骤是在良好的条件下隔离单个β细胞。充分灌注胰腺对后续消化反应较好。灌注不足, 通常发生在背胰, 将导致低胰岛产量。灌注后, 消化时间和晃动强度需要特别注意。过度消化, 由于长时间...

胰腺是哺乳动物重要的代谢器官。胰腺由内分泌和外分泌的隔间组成。胰腺内分泌细胞, 包括产生胰岛素的β细胞和胰高血糖素生成α细胞, 聚集在胰岛并协调调节系统葡萄糖稳态。内分泌细胞功能失调导致糖尿病, 这已成为世界上一个重要的公共卫生问题。
胰腺内分泌细胞在胚胎发生1期间由 Ngn3+祖祖先衍生。后期, 在围产期, 内分泌细胞增殖形成未成熟的胰岛。这些不成熟的细胞继续发展, 逐渐成为成熟的胰岛, 这成为丰富的血管化调节血糖动态平衡在成人2。
虽然已经确定了一组转录因子来调节β细胞分化, 但β细胞的精确成熟途径还不清楚。此外, β细胞的成熟过程也涉及到细胞数量扩展3,4和细胞异质性的生成5,6。然而, 这些过程的监管机制还没有得到很好的研究。
单细胞 RNA 测序是一种强有力的方法, 可以分析细胞亚群和跟踪细胞谱系发展途径7。利用这一技术, 在胰岛发育过程中发生的关键事件可以在单细胞8级破译出来。在单细胞 RNA 测序协议中, Smart-seq2 允许生成全长 cDNA, 提高灵敏度和准确度, 并使用标准试剂降低成本9。Smart-seq2 大约需要两天时间来构建一个用于序列10的 cDNA 文库。
在这里, 我们提出了一种方法, 从胎儿胰腺的荧光标记β细胞分离到成年 Ins1-RFP 转基因小鼠11, 使用荧光活化细胞分类 (资产管制署), 并在 transcriptomic 分析的性能单细胞水平, 使用 Smart-seq2 技术 (图 1)。该协议可推广到所有胰腺内分泌细胞类型在正常、病理和衰老状态下的 transcriptomes 分析。

<table>
	<tbody>
		<tr>
			<td>Collagenase P</td>
			<td>Roche</td>
			<td>11213873001</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.25 %), phenol red</td>
			<td>Thermo Fisher Scientific</td>
			<td>25200114</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Hyclone</td>
			<td>SH30071.03</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #4 Forceps</td>
			<td>Roboz</td>
			<td>RS-4904</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #5 Forceps</td>
			<td>Roboz</td>
			<td>RS-5058</td>
			<td></td>
		</tr>
		<tr>
			<td>30 G BD Needle 1/2&#34; Length</td>
			<td>BD</td>
			<td>305106</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereo Microscope</td>
			<td>Zeiss</td>
			<td>Stemi DV4</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereo Fluorescence microscope</td>
			<td>Zeiss</td>
			<td>Stereo Lumar V12</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>5810R</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>5424R</td>
			<td></td>
		</tr>
		<tr>
			<td>Polystyrene Round-Bottom Tube with Cell-Strainer Cap</td>
			<td>BD-Falcon</td>
			<td>352235</td>
			<td></td>
		</tr>
		<tr>
			<td>96-Well PCR Microplate</td>
			<td>Axygen</td>
			<td>PCR-96-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicone Sealing Mat</td>
			<td>Axygen</td>
			<td>AM-96-PCR-RD</td>
			<td></td>
		</tr>
		<tr>
			<td>Thin Well PCR Tube</td>
			<td>Extragene</td>
			<td>P-02X8-CF</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell sorter</td>
			<td>BD Biosciences</td>
			<td>BD FACSAria</td>
			<td></td>
		</tr>
		<tr>
			<td>Capillary pipette</td>
			<td>Sutter</td>
			<td>B100-58-10</td>
			<td></td>
		</tr>
		<tr>
			<td>RNaseZap</td>
			<td>Ambion</td>
			<td>AM9780</td>
			<td></td>
		</tr>
		<tr>
			<td>ERCC RNA Spike-In Mix</td>
			<td>Life Technologies</td>
			<td>4456740</td>
			<td></td>
		</tr>
		<tr>
			<td>Distilled water</td>
			<td>Gibco</td>
			<td>10977</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>T9284</td>
			<td></td>
		</tr>
		<tr>
			<td>dNTP mix</td>
			<td>New England Biolabs</td>
			<td>N0447</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant RNase Inhibitor</td>
			<td>Takara</td>
			<td>2313</td>
			<td></td>
		</tr>
		<tr>
			<td>Superscript II reverse transcriptase</td>
			<td>Invitrogen</td>
			<td>18064-014</td>
			<td></td>
		</tr>
		<tr>
			<td>First-strand buffer (5x)</td>
			<td>Invitrogen</td>
			<td>18064-014</td>
			<td></td>
		</tr>
		<tr>
			<td>DTT</td>
			<td>Invitrogen</td>
			<td>18064-014</td>
			<td></td>
		</tr>
		<tr>
			<td>Betaine</td>
			<td>Sigma-Aldrich</td>
			<td>107-43-7</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma-Aldrich</td>
			<td>7786-30-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Nuclease-free water</td>
			<td>Invitrogen</td>
			<td>AM9932</td>
			<td></td>
		</tr>
		<tr>
			<td>KAPA HiFi HotStart ReadyMix (2x)</td>
			<td>KAPA Biosystems</td>
			<td>KK2601</td>
			<td></td>
		</tr>
		<tr>
			<td>VAHTS DNA Clean Beads XP beads</td>
			<td>Vazyme</td>
			<td>N411-03</td>
			<td></td>
		</tr>
		<tr>
			<td>Qubit dsDNA HS Assay Kit</td>
			<td>Invitrogen</td>
			<td>Q32854</td>
			<td></td>
		</tr>
		<tr>
			<td>AceQ qPCR SYBR Green Master Mix</td>
			<td>Vazyme</td>
			<td>Q121-02</td>
			<td></td>
		</tr>
		<tr>
			<td>TruePrep DNA Library Prep Kit V2 for Illumina</td>
			<td>Vazyme</td>
			<td>TD502</td>
			<td>Include 5x TTBL, 5x TTE, 5x TS, 5x TAB, TAE</td>
		</tr>
		<tr>
			<td>TruePrep Index Kit V3 for Illumina</td>
			<td>Vazyme</td>
			<td>TD203</td>
			<td>Include 16 N6XX and 24 N8XX</td>
		</tr>
		<tr>
			<td>High Sensitivity NGS Fragment Analysis Kit</td>
			<td>Advanced Analytical Technologies</td>
			<td>DNF-474</td>
			<td></td>
		</tr>
		<tr>
			<td>1x HBSS without Ca2+ and Mg2+</td>
			<td></td>
			<td></td>
			<td>138 mM NaCl; 5.34 mM KCl 
			4.17 mM NaHCO3; 0.34 mM Na2HPO4 
			0.44 mM KH2PO4</td>
		</tr>
		<tr>
			<td>Isolation buffer</td>
			<td></td>
			<td></td>
			<td>1 &#215; HBSS containing 10 mM HEPES, 1 mM MgCl2, 5 mM Glucose, pH 7.4</td>
		</tr>
		<tr>
			<td>FACS buffer</td>
			<td></td>
			<td></td>
			<td>1 &#215; HBSS containing 15 mM HEPES, 5.6 mM Glucose, 1% FBS, pH 7.4</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>S5886</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma-Aldrich</td>
			<td>P9541</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Sigma-Aldrich</td>
			<td>S6297</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2HPO4</td>
			<td>Sigma-Aldrich</td>
			<td>S5136</td>
			<td></td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Sigma-Aldrich</td>
			<td>P5655</td>
			<td></td>
		</tr>
		<tr>
			<td>D-(+)-Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G5767</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H4034</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma-Aldrich</td>
			<td>M2393</td>
			<td></td>
		</tr>
		<tr>
			<td>Oligo-dT30VN primer</td>
			<td>5&#39;-AAGCAGTGGTATCAACGCAGAGTACT30VN-3&#39;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TSO</td>
			<td>5&#39;-AAGCAGTGGTATCAACGCAGAGTACATrGrG+G-3&#39;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ISPCR primers</td>
			<td>5&#39;-AAGCAGTGGTATCAACGCAGAGT-3&#39;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Gapdh Forward primer</td>
			<td>5&#39;-ATGGTGAAGGTCGGTGTGAAC-3&#39;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Gapdh Reverse primer</td>
			<td>5&#39;-GCCTTGACTGTGCCGTTGAAT-3&#39;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ins2 Forward primer</td>
			<td>5&#39;-TGGCTTCTTCTACACACCCA-3&#39;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ins2 Reverse primer</td>
			<td>5&#39;-TCTAGTTGCAGTAGTTCTCCA-3&#39;</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

single-cell transcriptomic analyses of mouse pancreatic endocrine cells

胰腺内分泌细胞聚集在胰岛, 调节血糖稳定性和能量代谢。胰岛细胞的不同类型, 包括胰岛素分泌β细胞, 在胚胎阶段与常见的内分泌祖先分化。未成熟的内分泌细胞通过细胞增殖和成熟在长时间的产后发育期内扩张。然而, 这些进程所依据的机制并没有明确界定。单细胞 RNA 测序是一种很有希望的方法来表征不同的细胞种群和追踪细胞谱系分化途径。在这里, 我们描述了从胚胎, 新生儿和产后胰腺分离的胰β细胞单细胞 RNA 序列的方法。

胰腺从胚胎, 新生儿和产后小鼠 (图 2A 和2B) 进行解剖。对于18岁以上的小鼠, 消化作用取决于灌注程度;因此, 注射是胰岛隔离的最重要步骤 (图 2C-2E和表 6)。在这一步中, 尽可能多地注射胶原酶来填充胰腺。完全膨胀的胰腺如图 2D所示。如果灌注不成功 (<st...

Watch this Scientific Journal Video about Single-cell Transcriptomic Analyses of Mouse Pancreatic Endocrine Cells at JoVE.com

Single-cell Transcriptomic Analyses of Mouse Pancreatic Endocrine Cells

我们描述了一种方法, 从胚胎, 新生儿和产后胰腺的内分泌细胞分离, 其次是单细胞 RNA 测序。这种方法可以分析胰腺内分泌谱系的发展, 细胞异质性和 transcriptomic 动力学。

小鼠胰腺内分泌细胞的单细胞 Transcriptomic 分析

Pancreatic endocrine cells, which are clustered in islets, regulate blood glucose stability and energy metabolism. The distinct cell types in islets, ...

该方法可以回答胰岛内关于胰岛血统分化、成熟和再生的关键问题。该技术的主要优点是，它允许收集单个胰腺胰岛细胞，以生成高质量的单细胞RNA测序数据。该技术的应用通过胰腺内分泌单细胞对病理条件的转录分析，应用于糖尿病和其他胰腺内分泌相关疾病的治疗。这种方法的视觉演示至关重要，因为将针头插入胰腺的狭窄胆管进行灌注可能比较棘手。对于胚胎日17.5胚胎。打开腹腔后，用肘钳将内脏组织转移到含有冷 PBS 的六厘米黑底盘。使用钳子将胰腺从内脏组织中分离出来。将胰腺组织放在含有5毫升冷胶原酶溶液的20毫升小瓶中。对于产后零至15只小鼠，在将组织放入胶原酶之前，请仔细解剖胰腺的所有叶。对于产后第18至60天小鼠，首先取出西腓，将肠道提取到腹腔右侧。将肝脏的左右中叶推到两侧，露出胆囊和普通胆管。在上部和下部位置用一对小容器夹住十二指肠，使胆管进入十二指肠的场地侧翼。接下来，加载一个5毫升注射器，配备30量针与冷胶原酶溶液。并使用显微镜将针尖插入胆囊。小心和平稳地通过普通胆管操纵针头。并按下柱塞，根据小鼠的大小，用一到五毫升的胶原酶溶液来使胰腺具有。当所有溶液都已注入时，使用钳子立即将胰腺转移到含有 5 毫升新鲜冷胶原蛋白溶液的 20 毫升小瓶中。收集完所有胰腺组织后，将小瓶放在37摄氏度的水浴中3分钟，然后轻轻摇晃3至5分钟。然后通过0.25毫米尼龙过滤器将消化的产品过滤到新的50毫升离心管中。并使用含有冰冷 PBS 的 20 毫升注射器彻底清洗过滤器。对于产后第 18 至 60 天胰腺，将过滤的样品离心机，并在 30 毫升冷 PBS 中重新暂停组织。然后将组织悬浮液放入六厘米的黑色底盘中，并使用 200 微升移液器和解剖显微镜将小岛拾取，以转移到含有少量冷 PBS 的 1.5 毫升微离心管中。对于胚胎期17.5产后第15胰腺，离心过滤的胰腺组织，并在胰腺素-EDTA溶液中重新暂停颗粒，在37摄氏度的水浴中孵育4分钟。在孵育结束时，用200微升移液器轻轻对颗粒进行一分钟的评价，然后加入0.5倍的冷胎牛血清体积，轻轻的涡流停止消化。然后通过离心收集消化液，并在5毫升的FACS管中将200微升的FACS缓冲液中重新填充，然后通过流式细胞分法进行分拣。对于单细胞采摘和解解，等分新鲜准备的细胞解液缓冲液分成8条PCR管，并将单个细胞转移到每个管中。涡流样品，使细胞释放RNA，随后在4摄氏度下离心30秒，并立即储存在冰上。对于逆转录，在72摄氏度下孵育3分钟，随后在冰上孵育1分钟。在4摄氏度下将样品短暂离心30秒，并将5.7微升的逆转录混合物添加到每个样品中，使每个样品的总体积达到10微升。温和涡流和离心后，将样品装入热循环器，然后开始逆转录程序。对于聚合酶链式反应预增，或 PCR，在包含第一股反应的每个样品中加入 15 微升 PCR 混合物，然后进行温和涡流和离心。然后将样品加载到热循环器中，然后启动 PCR 程序。对于 PCR 纯化，在每个样品中加入 25 微升的重新暂停的 DNA 纯化珠，通过涡流混合良好。然后在室温下快速旋转样品以收集液体，同时避免珠子沉降。在室温下五分钟后，将样品放在适当的磁性支架上，在溶液清除时小心地取出和丢弃上清液。用两个30秒洗涤珠子，在200微升新鲜准备的80%乙醇每次洗涤。第二次洗涤后丢弃剩余的乙醇。当珠子风干时，加入11微升无核酸水，从珠子中检测出DNA靶点，然后进行涡流。然后快速离心样品，将样品返回磁站，直到溶液清除，将每个样品的10微升转移到新的PCR管中。成功放大的cDNA应具有500个基对以上的全长，并丰富从1.5至2个千斤基对。此外，在胰岛素中观察到的cDNA基对浓缩有500至600基对，一个RFP加细胞，这可能代表胰岛素记录。然而，100个碱基对附近的cDNA片段是底像二丁二线，通常由过多的底能引起，必须通过重复DNA纯化步骤去除。100 到 500 个基对之间的 cDNA 片段通常表示降解的 cDNA，这是由不良细胞状态或区域问题（如 RNase 污染）引起的，应排除。在cDNA库进行测序纯化后，可以通过在第一轮和第二轮纯化中加入DNA纯化珠获得250至450个碱基对的cDNA。DNA珠纯化后，测序质量评价中测序质量得分应大于30。为了获得用于下游分析的高质量细胞，排除了读取量小于0.5万或检测到少于4000个基因的细胞，从而有助于不同细胞群的表征，以及在主要成分分析和聚类分层后，识别不同组异构表达的基因。在尝试此程序时，重要的是要记住在胶原酶消化之前快速吸收胰腺，以避免胰岛过度消化，并在单细胞程序期间保持无 RNase 环境。按照这个程序，其他方法，如小岛成像和文化可以执行，以促进小岛结构的可视化，并检查细胞反应和药物刺激。开发后，该技术为胰腺研究领域的研究人员探索胰岛血统发育和再生机制以及哺乳动物糖尿病进展铺平了道路。

Research

JoVE Journal

Developmental Biology

Efficient Differentiation of Pluripotent Stem Cells to NKX6-1+ Pancreatic Progenitors

Efficient Differentiation of Pluripotent Stem Cells to NKX6-1+ Pancreatic Progenitors

多能高效分化干细胞NKX6-1<sup&gt; +</sup&gt;胰腺祖

Pluripotent stem cells have the ability to self renew and differentiate to multiple lineages, making them an attractive source for the generation of ...

科研

发育生物学

Single-cell Photoconversion in Living Intact Zebrafish

活完好斑马鱼的单细胞 Photoconversion

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause ...

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

无支架三维胰岛素表达 Pancreatoids 的实验研究体外培养的小鼠胰祖细胞

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

秀丽线虫生殖的计算分析研究细胞核、蛋白质和细胞骨架的分布

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

人诱导多能干细胞体外生成体细胞衍生物的研究

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...

Methods to Test Endocrine Disruption in Drosophila melanogaster

Methods to Test Endocrine Disruption in Drosophila melanogaster

检测果蝇黑色素的内分泌干扰的方法

In recent years there has been growing evidence that all organisms and the environment are exposed to hormone-like chemicals, known as endocrine disruptor ...

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C (UV-C) Damage

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C UV-C Damage

评估原小鼠眼部表面细胞/干细胞对紫外线-C （UV-C） 损伤的反应

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational ...

Efficient Neural Differentiation using Single-Cell Culture of Human Embryonic Stem Cells

利用人类胚胎干细胞的单细胞培养进行高效神经分化

In vitro differentiation of human embryonic stem cells (hESCs) has transformed the ability to study human development on both biological and molecular ...

Multiplexed Single Cell mRNA Sequencing Analysis of Mouse Embryonic Cells

小鼠胚胎细胞的多路复用单细胞mRNA测序分析

Single cell mRNA sequencing has made significant progress in the last several years and has become an important tool in the field of developmental ...

Preparation of Small RNA Libraries for Sequencing from Early Mouse Embryos

为早期小鼠胚胎测序准备小型RNA库

MicroRNAs (miRNAs) are important for the complex regulation of cell fate decisions and developmental timing. In vivo studies of the contribution of miRNAs ...