Identification of key factors regulating self-renewal and differentiation in EML hematopoietic precursor cells by RNA-sequencing analysis.
Identification of key factors regulating self-renewal and differentiation in EML hematopoietic precursor cells by RNA-sequencing analysis.
复制标题
DOI：
10.3791/52104
复制
发表时间：
2014-11-11
期刊：
影响因子：
--
通讯作者：
Wu JQ
中科院分区：
文献类型：
--
作者：
Zong S;Deng S;Chen K;Wu JQ
造血干细胞（HSC）在临床上用于移植治疗白血病、淋巴瘤等多种疾病，以重建患者的造血系统。阐明控制 HSC 自我更新和分化的机制对于 HSC 的研究和临床应用具有重要意义。然而，由于HSCs无法在体外增殖，因此不可能获得大量的HSCs。为了克服这一障碍，我们使用小鼠骨髓来源的细胞系 EML（红细胞、髓细胞和淋巴细胞）细胞系作为本研究的模型系统。 RNA 测序 (RNA-Seq) 已越来越多地用于替代基因表达研究的微阵列。我们在这里报告了一种使用RNA-Seq技术来研究EML细胞自我更新和分化调节的潜在关键因素的详细方法。本文提供的协议分为三个部分。第一部分解释如何培养 EML 细胞并分离 Lin-CD34+ 和 Lin-CD34− 细胞。该协议的第二部分提供了总 RNA 制备和随后的高通量测序文库构建的详细程序。最后一部分描述了RNA-Seq数据分析的方法，并解释了如何使用数据来识别Lin-CD34+和Lin-CD34−细胞之间差异表达的转录因子。差异表达最显着的转录因子被确定为控制 EML 细胞自我更新和分化的潜在关键调节因子。在本文的讨论部分，我们重点介绍了成功进行该实验的关键步骤。总之，本文提供了一种利用RNA-Seq技术来识别EML细胞自我更新和分化的潜在调节因子的方法。确定的关键因素将在体外和体内进行下游功能分析。
Hematopoietic stem cells (HSCs) are used clinically for transplantation treatment to rebuild a patient's hematopoietic system in many diseases such as leukemia and lymphoma. Elucidating the mechanisms controlling HSCs self-renewal and differentiation is important for application of HSCs for research and clinical uses. However, it is not possible to obtain large quantity of HSCs due to their inability to proliferate in vitro. To overcome this hurdle, we used a mouse bone marrow derived cell line, the EML (Erythroid, Myeloid, and Lymphocytic) cell line, as a model system for this study. RNA-sequencing (RNA-Seq) has been increasingly used to replace microarray for gene expression studies. We report here a detailed method of using RNA-Seq technology to investigate the potential key factors in regulation of EML cell self-renewal and differentiation. The protocol provided in this paper is divided into three parts. The first part explains how to culture EML cells and separate Lin-CD34+ and Lin-CD34− cells. The second part of the protocol offers detailed procedures for total RNA preparation and the subsequent library construction for high-throughput sequencing. The last part describes the method for RNA-Seq data analysis and explains how to use the data to identify differentially expressed transcription factors between Lin-CD34+ and Lin-CD34− cells. The most significantly differentially expressed transcription factors were identified to be the potential key regulators controlling EML cell self-renewal and differentiation. In the discussion section of this paper, we highlight the key steps for successful performance of this experiment. In summary, this paper offers a method of using RNA-Seq technology to identify potential regulators of self-renewal and differentiation in EML cells. The key factors identified are subjected to downstream functional analysis in vitro and in vivo.
登录
查看更多内容
影响因子：
--
作者：
Bolduc F;Hoareau C;St-Pierre P;Perreault JP
通讯作者：
Perreault JP
DOI：
10.1093/bioinformatics/btq033
发表时间：
2010-03-15
期刊：
Bioinformatics (Oxford, England)
影响因子：
--
作者：
Quinlan AR;Hall IM
通讯作者：
Hall IM
影响因子：
3
作者：
Sun L;Zhang Z;Bailey TL;Perkins AC;Tallack MR;Xu Z;Liu H
通讯作者：
Liu H
影响因子：
46.9
作者：
Trapnell C;Williams BA;Pertea G;Mortazavi A;Kwan G;van Baren MJ;Salzberg SL;Wold BJ;Pachter L
通讯作者：
Pachter L
影响因子：
2.1
作者：
Murphy, D
通讯作者：
Murphy, D