Puf proteins, RNA co-immunopurification

RNA-coimmunopurifications with TAP-tagged Puf proteins from Saccharomyces cereviseae. Untagged strain (BY4741) served as a control. Cells were grown to midlog phase and harvested by centrifugation. TAP-tagged Puf proteins were affinity purified from cell-free extracts with IgG sepharose and eluted with TEV protease. RNA was isolated from extract (=input)and from purified protein samples by phenol-chloroform extraction. RNA samples were reverse transcribed using a mixture of oligo-dT and random nonamer oligos in the presence of amino-allyl dUTP/ dNTP mixture. cDNAs were fluorescently labeled and hybridized on yeast DNA microarrays over night at 65 degrees. For a detailed procedure see http://microarray-pubs.stanford.edu/yeast_puf and also Gerber AP et al. PLoS Biology, 2004. Set of arrays organized by shared biological context, such as organism, tumors types, processes, etc. Keywords: Logical Set Computed

本数据集针对酿酒酵母（Saccharomyces cereviseae）中TAP标记的Puf蛋白开展RNA免疫共沉淀（RNA-coimmunopurification）实验，以未携带标签的BY4741菌株作为对照。将细胞培养至对数中期后通过离心收集菌体，从无细胞提取物中利用IgG琼脂糖磁珠亲和纯化TAP标记的Puf蛋白，并通过TEV蛋白酶（TEV protease）洗脱。采用酚-氯仿抽提法，分别从提取物（即输入样本）与纯化的蛋白样本中分离RNA。以寡聚dT（oligo-dT）与随机九聚寡核苷酸的混合引物，在氨基烯丙基dUTP/dNTP混合液存在的条件下对RNA样本进行逆转录，将所得cDNA进行荧光标记后，于65℃下在酵母DNA微阵列（DNA microarray）上进行过夜杂交。详细实验流程可参见http://microarray-pubs.stanford.edu/yeast_puf 及Gerber AP等人发表于《公共科学图书馆·生物学》2004年的研究论文。本微阵列数据集按照共享的生物学背景进行归类，例如物种、肿瘤类型、生物学过程等。关键词：已计算逻辑集