Transcriptional profiling of hematopoietic stem cells by high-throughput sequencing
Transcriptional profiling of hematopoietic stem cells by high-throughput sequencing
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DOI：
10.1007/s12185-008-0212-2
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发表时间：
2009-01
影响因子：
2.1
通讯作者：
Yoshimi Yashiro;H. Bannai;T. Minowa;Tomohide Yabiku;S. Miyano;M. Osawa;A. Iwama;H. Nakauchi
中科院分区：
文献类型：
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作者：
Yoshimi Yashiro;H. Bannai;T. Minowa;Tomohide Yabiku;S. Miyano;M. Osawa;A. Iwama;H. Nakauchi
微阵列分析使得在一次检测中进行广泛的基因表达谱分析成为可能。各种造血干细胞（HSC）群体已经进行了微阵列分析和他们的基因表达谱已被报道。然而，这种方法不适合鉴定新的转录本或用于低表达水平基因的分析。为了获得CD 34 −c-Kit+Sca-1+谱系标记阴性（Lin−）（CD 34 −KSL）HSC的详细基因表达谱，我们构建了CD 34 −KSL cDNA文库，进行了高通量测序，并将生成的谱与另一种HSC组分，侧群（SP）Lin−（SP Lin−）细胞的谱进行了比较。对来自每个HSC文库的约9，500个cDNA的5′-末端进行测序，分别从CD 34 −KSL和SP Lin−文库中鉴定出1，424和2，078个不同的基因。为了排除包括管家基因在内的广泛表达的基因，成功地对其他器官的EST数据库进行了数字减法，留下了25个HSC特异性基因，包括5个新基因。在来自CD 34 −KSL cDNA文库的4，450个转录物中，显示与推定的蛋白质编码基因没有同源性，通过计算机分析，29个被鉴定为mRNA样非编码RNA的强候选者。我们的cyclopedic方法可能有助于理解新的HSC功能的分子方面。
Microarray analysis has made it feasible to carry out extensive gene expression profiling in a single assay. Various hematopoietic stem cell (HSC) populations have been subjected to microarray analyses and their profiles of gene expression have been reported. However, this approach is not suitable to identify novel transcripts or for profiling of genes with low expression levels. To obtain a detailed gene expression profile of CD34−c-Kit+Sca-1+lineage marker-negative (Lin−) (CD34−KSL) HSCs, we constructed a CD34−KSL cDNA library, performed high-throughput sequencing, and compared the generated profile with that of another HSC fraction, side population (SP) Lin−(SP Lin−) cells. Sequencing of the 5′-termini of about 9,500 cDNAs from each HSC library identified 1,424 and 2,078 different genes from the CD34−KSL and SP Lin−libraries, respectively. To exclude ubiquitously expressed genes including housekeeping genes, digital subtraction was successfully performed against EST databases of other organs, leaving 25 HSC-specific genes including five novel genes. Among 4,450 transcripts from the CD34−KSL cDNA library that showed no homology to the presumable protein-coding genes, 29 were identified as strong candidates for mRNA-like non-coding RNAs by in silico analyses. Our cyclopedic approaches may contribute to understanding of novel molecular aspects of HSC function.