Wellcome Trust/Cancer Research UK Gurdon Institute of Cancer and Developmental Biology, University of Cambridge, Cambridge, UK.
Nat Protoc. 2010 Mar;5(3):516-35. doi: 10.1038/nprot.2009.236. Epub 2010 Feb 25.
We describe here a protocol for digital transcriptome analysis in a single mouse oocyte and blastomere using a deep-sequencing approach. In this method, individual cells are isolated and transferred into lysate buffer by mouth pipette, followed by reverse transcription carried out directly on the whole cell lysate. Free primers are removed by exonuclease I and a poly(A) tail is added to the 3' end of the first-strand cDNAs by terminal deoxynucleotidyl transferase. Single-cell cDNAs are then amplified by 20 + 9 cycles of PCR. The resulting 100-200 ng of amplified cDNAs are used to construct a sequencing library, which can be used for deep sequencing using the SOLiD system. Compared with cDNA microarray techniques, our assay can capture up to 75% more genes expressed in early embryos. This protocol can generate deep-sequencing libraries for 16 single-cell samples within 6 d.
我们在这里描述了一种使用深度测序方法在单个小鼠卵母细胞和胚胎中进行数字转录组分析的方案。在该方法中,通过移液管逐个分离细胞并转移到裂解缓冲液中,然后直接在整个细胞裂解物上进行逆转录。通过外切核酸酶 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 poly(A) 尾巴。然后通过 20+9 个循环的 PCR 扩增单细胞 cDNA。所得的 100-200ng 扩增 cDNA 用于构建测序文库,可用于 SOLiD 系统的深度测序。与 cDNA 微阵列技术相比,我们的检测方法可以捕获多达 75%的早期胚胎中表达的基因。该方案可在 6 天内为 16 个单细胞样本生成深度测序文库。