RNA-Seq analysis to capture the transcriptome landscape of a single cell.
RNA-Seq analysis to capture the transcriptome landscape of a single cell.
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DOI：
10.1038/nprot.2009.236
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发表时间：
2010-03
期刊：
影响因子：
14.8
通讯作者：
中科院分区：
文献类型：
--
作者：
我们在此描述一种使用深度测序方法对单个小鼠卵裂球进行数字转录组分析的实验方案。首先用口吸管分离单个卵裂球并将其置于裂解缓冲液中。然后直接在全细胞裂解物上进行逆转录。在此之后，用核酸外切酶I去除游离引物，并通过末端脱氧核苷酸转移酶在第一链cDNA的3′端添加多聚（A）尾。然后通过20 + 9个PCR循环扩增单细胞cDNA。接着使用100 - 200纳克这些扩增的cDNA构建测序文库。该测序文库可用于SOLiD系统进行深度测序。与cDNA微阵列技术相比，我们的检测方法能够多捕获早期胚胎中多达75%的表达基因。该实验方案能够在6天内为16个单细胞样本生成深度测序文库。
We describe here a protocol for digital transcriptome analysis in a single mouse blastomere using a deep sequencing approach. An individual blastomere was first isolated and put into lysate buffer by mouth pipette. Reverse transcription was then performed directly on the whole cell lysate. After this, the free primers were removed by Exonuclease I and a poly(A) tail was added to the 3′ end of the first-strand cDNA by Terminal Deoxynucleotidyl Transferase. Then the single cell cDNAs were amplified by 20 plus 9 cycles of PCR. Then 100-200 ng of these amplified cDNAs were used to construct a sequencing library. The sequencing library can be used for deep sequencing using the SOLiD system. Compared with the cDNA microarray technique, our assay can capture up to 75% more genes expressed in early embryos. The protocol can generate deep sequencing libraries within 6 days for 16 single cell samples.
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影响因子：
23.9
作者：
Hayashi K;de Sousa Lopes SMC;Tang F;Lao K;Surani MA
通讯作者：
Surani MA
影响因子：
48
作者：
Cloonan, Nicole;Forrest, Alistair R. R.;Grimmond, Sean M.
通讯作者：
Grimmond, Sean M.
DOI：
10.1093/bfgp/2.1.31
发表时间：
2003-04-01
期刊：
Briefings in Functional Genomics & Proteomics
影响因子：
--
作者：
Livesey, F. J.
通讯作者：
Livesey, F. J.
影响因子：
14.9
作者：
Hartmann, Claudia H.;Klein, Christoph A.
通讯作者：
Klein, Christoph A.
影响因子：
14.8
作者：
Kurimoto, Kazuki;Yabuta, Yukihiro;Saitou, Mitinori
通讯作者：
Saitou, Mitinori