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Author:
Date:
2013-06-20
[Abstract] High throughput RNA Sequencing has revolutionized transcriptome analyses. However, most available protocols require micrograms of RNA rendering this technique not feasible for analyzing small numbers of cells, including precious rare cell types isolated from human tissues or organs. Here, we used an RNA Amplification System and describe a method for preparing RNA sense-strand cDNA libraries compatible with an Illumina sequencing platform starting from limited numbers of human fetal germ cells as well as human embryonic stem cells (hESCs) isolated using Fluorescence Activated Cell Sorting (FACS). With this protocol we generated seven RNA-Seq libraries starting from 4,000 germ cells sorted from fetal ovaries (n = 2) and fetal testes (n = 2) at 16-16.5 weeks of development and 4,000 sorted ...
[摘要] 高通量RNA测序革新了转录组分析。 然而，大多数可用的协议需要微克RNA，使得这种技术不可能用于分析少量的细胞，包括从人体组织或器官分离的珍贵的稀有细胞类型。 在这里，我们使用RNA扩增系统，并描述了一种制备RNA有义链cDNA文库的方法，其与Illumina测序平台相容，从有限数目的人胎儿生殖细胞以及使用荧光活化细胞分离的人胚胎干细胞（hESC） 排序（FACS）。 使用这个协议，我们生成七个RNA Seq库开始从4,000生殖细胞从胎儿卵巢（n = 2）和胎儿睾丸（n = 2）在16-16.5周的发展和4,000分选hESCs（n = 3）排序。 我们预测多重文库也可以通过用多重兼容的3'衔接子和索引的PCR引物替换这里使用的单重3'衔接子来产生。
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