CLIP-seq of Salmonella ProQ in intracellular-like conditions

The aim of the project is to identify transcriptome-wide binding sites for the global RNA-binding protein ProQ in Salmonella during intracellular-like conditions. Salmonella enterica strain SL1344 carrying a chromosomal proQ-3xFLAG allele was grown in intracellular-like growth medium until and OD600 of 0.3 and thereafter subjected to crosslinking immunoprecipitation sequencing (CLIP-seq). Specifically, the bacteria were subjected to UV-light-induced crosslinking, anti-FLAG-mediated immunoprecipitation, RNase treatment, gel purification, and RNA purification, followed by Illumina library preparation using the NEBNext Small RNA Prep kit and sequencing on an Illumina NextSeq500 instrument at vertis Biotechnologie AG (Freising, Germany).

本项目旨在模拟胞内环境条件下，鉴定肠炎沙门氏菌（Salmonella enterica）中全局RNA结合蛋白（RNA-binding protein）ProQ的全转录组结合位点。携带染色体上proQ-3xFLAG等位基因的肠炎沙门氏菌SL1344菌株，在模拟胞内生长培养基中培养至OD600值为0.3，随后进行交联免疫沉淀测序（crosslinking immunoprecipitation sequencing，CLIP-seq）。具体而言，先对细菌进行紫外光诱导交联、抗FLAG抗体介导的免疫沉淀、核糖核酸酶（RNase）处理、凝胶纯化与RNA纯化（RNA purification）；随后使用NEBNext Small RNA Prep试剂盒进行Illumina测序文库制备，并于德国弗赖辛的vertis Biotechnologie AG公司的Illumina NextSeq500测序仪上完成测序。