Mouse kidney nuclear isolation and library preparation for single-cell combinatorial indexing RNA sequencing.
Mouse kidney nuclear isolation and library preparation for single-cell combinatorial indexing RNA sequencing.
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DOI：
10.1016/j.xpro.2022.101904
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发表时间：
2022-12-16
期刊：
影响因子：
--
通讯作者：
Humphreys, Benjamin D.
中科院分区：
文献类型：
--
作者：
Li, Haikuo;Humphreys, Benjamin D.
单细胞组合索引RNA测序（sci-RNA-seq 3）能够在一个实验中对多个样品进行高通量单核转录组学分析。在这里，我们描述了一种优化的小鼠肾细胞核分离和sci-RNA-seq 3文库制备方案。使用dounce组织匀浆器能够以高产率提取细胞核。将固定的核处理用于sci-RNA-seq 3，并将自加载的转座体Tn 5用于文库生成中的标签片段化。逐步的协议允许研究人员以低成本使用常见的实验室用品生成可扩展的单细胞转录组数据。有关本方案使用和执行的完整详细信息，请参见Li等人。（2022年）。优化的小鼠肾脏细胞核分离方案，使用退火寡核苷酸进行高效Tn 5组装，以进行功能性转座体转座体活性滴定测试，以确定最佳工作浓度执行小规模sci-RNA-seq 3实验作为原理证明出版商说明：进行任何实验方案都需要遵守当地机构的实验室安全和伦理指南。单细胞组合索引RNA测序（sci-RNA-seq 3）能够在一个实验中对多个样品进行高通量单核转录组学分析。在这里，我们描述了一种优化的小鼠肾细胞核分离和sci-RNA-seq 3文库制备方案。使用dounce组织匀浆器能够以高产率提取细胞核。将固定的核处理用于sci-RNA-seq 3，并将自加载的转座体Tn 5用于文库生成中的标签片段化。逐步的协议允许研究人员以低成本使用常见的实验室用品生成可扩展的单细胞转录组数据。
Single-cell combinatorial indexing RNA sequencing (sci-RNA-seq3) enables high-throughput single-nucleus transcriptomic profiling of multiple samples in one experiment. Here, we describe an optimized protocol of mouse kidney nuclei isolation and sci-RNA-seq3 library preparation. The use of a dounce tissue homogenizer enables nuclei extraction with high yield. Fixed nuclei are processed for sci-RNA-seq3, and self-loaded transposome Tn5 is used for tagmentation in library generation. The step-by-step protocol allows researchers to generate scalable single-cell transcriptomic data with common laboratory supplies at low cost. For complete details on the use and execution of this protocol, please refer to Li et al. (2022). Optimized nuclei isolation protocol for mouse kidneys with high efficiency Tn5 assembly with annealed oligonucleotides to make functional transposome Transposome activity titration test to determine optimal working concentration Performing a small-scale sci-RNA-seq3 experiment as a proof of principle Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Single-cell combinatorial indexing RNA sequencing (sci-RNA-seq3) enables high-throughput single-nucleus transcriptomic profiling of multiple samples in one experiment. Here, we describe an optimized protocol of mouse kidney nuclei isolation and sci-RNA-seq3 library preparation. The use of a dounce tissue homogenizer enables nuclei extraction with high yield. Fixed nuclei are processed for sci-RNA-seq3, and self-loaded transposome Tn5 is used for tagmentation in library generation. The step-by-step protocol allows researchers to generate scalable single-cell transcriptomic data with common laboratory supplies at low cost.
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影响因子：
14.8
作者：
Martin, Beth K.;Qiu, Chengxiang;Nichols, Eva;Phung, Melissa;Green-Gladden, Rula;Srivatsan, Sanjay;Blecher-Gonen, Ronnie;Beliveau, Brian J.;Trapnell, Cole;Cao, Junyue;Shendure, Jay
通讯作者：
Shendure, Jay
影响因子：
64.8
作者：
Cao, Junyue;Spielmann, Malte;Shendure, Jay
通讯作者：
Shendure, Jay
影响因子：
16.6
作者：
Zheng GX;Terry JM;Belgrader P;Ryvkin P;Bent ZW;Wilson R;Ziraldo SB;Wheeler TD;McDermott GP;Zhu J;Gregory MT;Shuga J;Montesclaros L;Underwood JG;Masquelier DA;Nishimura SY;Schnall-Levin M;Wyatt PW;Hindson CM;Bharadwaj R;Wong A;Ness KD;Beppu LW;Deeg HJ;McFarland C;Loeb KR;Valente WJ;Ericson NG;Stevens EA;Radich JP;Mikkelsen TS;Hindson BJ;Bielas JH
通讯作者：
Bielas JH
DOI：
10.3791/3564
发表时间：
2012-07-30
期刊：
Journal of visualized experiments : JoVE
影响因子：
--
作者：
Gage, Gregory J;Kipke, Daryl R;Shain, William
通讯作者：
Shain, William
影响因子：
29
作者：
Li, Haikuo;Dixon, Eryn E.;Wu, Haojia;Humphreys, Benjamin D.
通讯作者：
Humphreys, Benjamin D.