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题目:: Affinity purification of ribosomes and associated RNAs using tagged Rpl16a and Rpl16b
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状态:: 发布时间June 25, 2010 , 更新时间 May 1, 2014 , 提交时间 Nov. 18, 2008,
物种:: Saccharomyces cerevisiae
摘要:: In this study, we systematically identified RNAs associated with ribosomes. To identify ribosome associated RNAs, C-terminal ZZ-tagged Rpl16a or Rpl16b, expressed under control of thier native promoter, were affinity purified from whole cell extracts of cultures grown to mid-log phase in minimal medium. Extracts were incubated with immunoglobulin G (IgG) coupled microbeads, washed, and ribosomes were eluted by tobacco etch virus (TEV) protease treatment. We performed two biological replicates with each protein and analyzed the RNA content using oligo microarrays. Total RNA isolated from extracts of cells expressing Rpl16a-ZZ or Rpl16b-ZZ (input) and from the affinity-purified ribosomes was reverse transcribed with oligo(dT) primers. cDNA was labeled with Cy3 and Cy5 fluorescent dyes, respectively, and competitively hybridized to yeast oligo microarrays. Alternatively, RNA was isolated from sucrose gradient fractions containing 60S subunits, 80S monosomes and polysomes. Here, we performed four biological replicates. Analysis was done with oligo microarrays as described above. A strain or line experiment design type assays differences between multiple strains, cultivars, serovars, isolates, lines from organisms of a single species. strain_or_line_design
实验种类:: other
样本量:: 16
实验设计:
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数据号:: E-GEOD-13654, GSE13654
数据状态:: 无法自动分析，您可以尝试手动分析数据。

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