miRNA expression in LT-HSCs upon Thrombopoietin stimulation via STAT5

<strong>File description:<br></strong> <strong><br></strong> <strong>MX: STAT5-deficient</strong> <strong>FL: STAT5-wt</strong> <strong>1/2: cohort 1 and 2</strong> <strong>+: TPO-stimulated</strong> <strong>-: unstimulated<br></strong> <strong><br></strong> <strong>MiRNA expression in murine long-term repopulating hematopoietic stem cells upon Thrombopoietin stimuation via STAT5A/B</strong> Conditional deletion of STAT5A/B was induced in 6-week old mice (STAT5fl/fl x Mx1:Cre and STAT5fl/fl; 16-20 mice per group and experiment) via Poly(I:C) injections as previously described. LT-HSCs were isolated from 12-16 week old mice via FACS sorting. 5000-10000 viable LT-HSCs were starved for 5h and subsequently stimulated wit 100ng/ml TPO (Peprotech) for 2h. The RNA was isolated by using a miRNeasy mini kit (Qiagen). cDNA was synthesised from total RNA using a microRNA gene-specific RT-primer pool according to the MicroRNA Megaplex Assay protocol (Megaplex™ Pools - Applied Biosystems). The preamplification reaction was performed according to the manufacture´s protocol. The TaqMan Array MicroRNA Rodent Cards were analysed using the 384 well TaqMan Low Density Array default thermal cycling conditions (ABI PRISM 7900HT).

<strong>文件说明：<br></strong> <strong><br></strong> <strong>MX：STAT5缺陷型（STAT5-deficient）</strong> <strong>FL：STAT5野生型（STAT5-wt）</strong> <strong>1/2：队列1与队列2</strong> <strong>+：经血小板生成素（TPO）刺激</strong> <strong>-：未刺激<br></strong> <strong><br></strong> <strong>经STAT5A/B介导的血小板生成素刺激后小鼠长期造血重建造血干细胞的微小RNA（miRNA）表达特征</strong> 本研究通过聚肌胞苷酸（Poly(I:C)）注射，对6周龄小鼠（每组每轮实验纳入16-20只小鼠，基因型为STAT5fl/fl × Mx1:Cre 与 STAT5fl/fl）诱导STAT5A/B条件性敲除，实验操作参照既往已发表方案。从12-16周龄小鼠中通过荧光激活细胞分选（FACS）分离长期造血重建造血干细胞（LT-HSCs）。取5000~10000个活的LT-HSCs进行5小时饥饿培养，随后以100ng/ml的血小板生成素（Peprotech公司产品）刺激2小时。采用miRNeasy微型试剂盒（Qiagen）提取总RNA，参照MicroRNA Megaplex检测试剂盒（Applied Biosystems公司Megaplex™ 混合池）的操作流程，使用微小RNA基因特异性逆转录引物池从总RNA中合成cDNA。按照制造商说明书完成预扩增反应，采用384孔TaqMan低密度芯片默认热循环条件（ABI PRISM 7900HT仪器）对TaqMan Array MicroRNA啮齿类芯片进行数据分析。