Next Generation Sequencing Facilitates Quantitative Analysis of RNAs bound by DDX5 and RORgt in cultured T helper 17 cell [RIP-seq]

Purpose: The goals of this study are to compare RNAs bound by DDX5 and RORgt in cultured T helper 17 cell in wildtype background. Methods: Th17 mRNA profiles of 48hrs in vitro cultured T helper 17 cells from wild-type mice were generated by deep sequencing, using Illumina HighSeq. The sequence reads that passed quality filters were analyzed at the transcript isoform level with two methods: Burrows–Wheeler Aligner (BWA) and TopHat followed by CuffDiff. qRT–PCR validation was performed using SYBR Green assays. Results: Among the 3444 RefSeq non-coding RNAs and 46449 NONCODE non-coding RNAs, 2533 were found to be expressed in Th17 cells (FPKM>1). 210 of them were enriched in DDX5 pull-down and 119 of them were enriched in RORgt pull-down. Conclusions: Our study suggest that a subset of 31 ncRNAs were co-enriched in DDX5 and RORgt pull-down. DDX5 or RORgt-associated-RNA profiles of 48hr in vitro cultured Th17 from wild type (WT) mice were generated by deep sequencing using Illumina HighSeq

研究目的：本研究旨在比较野生型背景下体外培养的辅助性T细胞17（T helper 17 cell, Th17）中被DDX5与RORgt结合的RNA分子。
实验方法：采用Illumina HighSeq平台对野生型小鼠来源的、体外培养48小时的Th17细胞进行深度测序，以获取其mRNA表达谱。对通过质量过滤的序列读段，分别采用两种方法在转录本异构体层面开展分析：Burrows–Wheeler Aligner（BWA）以及TopHat联合CuffDiff。采用SYBR Green检测法完成定量实时聚合酶链反应（qRT–PCR）验证实验。
实验结果：在3444条参考序列数据库（RefSeq）非编码RNA以及46449条NONCODE非编码RNA数据库收录的非编码RNA中，共鉴定出2533条在Th17细胞中表达（FPKM>1）。其中210条在DDX5下拉实验（pull-down）中富集，119条在RORgt下拉实验中富集。
研究结论：本研究表明，存在31个非编码RNA子集在DDX5与RORgt下拉实验中共同富集。本研究同时生成了野生型（WT）小鼠来源的、体外培养48小时的Th17细胞中DDX5或RORgt相关RNA谱的深度测序数据。