Trf4p in vivo crosslinking and ribonucleoprotein-immunopurification-chip analysis (X-RIP-Chip). Saccharomyces cerevisiae

In vivo cross-linking and ribonucleoprotein-immunopurification experiments followed by microarray analysis of bound RNAs (X-RIP-chip). Cells expressing recombinant tandem-affinity purification (TAP)-tagged Trf4 protein were cross-linked with formaldehyde, and Trf4-containing ribonucleoprotein complexes were recovered by affinity selection on IgG-coupled beads (see linked protocol). As a control for non-specifically enriched RNAs, the same experiment was done with untagged WT cells and with cells expressing Fpr1-TAP, a peptidyl-prolyl-cis-trans-isomerase not expected to bind RNA. Set of arrays organized by shared biological context, such as organism, tumors types, processes, etc. Antigenic peptide used in IP: Protein A derivative Overall design: Computed

本数据集采用体内交联（in vivo cross-linking）联合核糖核蛋白免疫纯化实验，并对结合的RNA进行微阵列分析（X-RIP-chip）。将表达重组串联亲和纯化（tandem-affinity purification, TAP）标签融合Trf4蛋白的细胞经甲醛交联后，通过IgG偶联磁珠（IgG-coupled beads）的亲和筛选回收含有Trf4的核糖核蛋白复合物（详见关联实验方案）。为设置非特异性富集RNA的对照，本研究分别以未标记野生型细胞、表达Fpr1-TAP的细胞开展相同实验；其中Fpr1-TAP为一种预期不会结合RNA的肽基脯氨酰顺反异构酶（peptidyl-prolyl-cis-trans-isomerase）。本数据集的芯片阵列按共享的生物学背景进行分组，涵盖生物体、肿瘤类型、生物学过程等类别。免疫沉淀（immunoprecipitation, IP）所用抗原肽为蛋白A衍生物（Protein A derivative）。整体实验设计：经计算得到。