Genome wide RNA sequencing to profile response to IL-1 in control and IFT88 depleted cells

Purpose: RNA sequencing across whole genome downstream to IL-1 to investigate scope and nature of effect of IFT88 depletion Method: Control pool treated and IFT88 siRNA pool targeted Fibroblast-like chondrocytes were treated with or without IL-1B 10ng/ml and RNA collected in triplicate at 0, 1, 2, 4, 8 and 24h. PolyA-selected sequencing libraries were prepared using the TruSeq protocol (Illumina). Libraries were subject to 75bp paired-end sequencing (Illumina HiSeq 4000) to an average depth of 28.5 million read pairs per sample and aligned to mouse genome with Hisat2. Results: Using time-course analysis (ImpulseDE2) the IL-1 response timecourse was determined (control-only) and intersected against the comparative case vs control analysis. Per-point differential expression analysis IFT88 vs control was performed using DESeq2. Conclusion: Demonstration of the effect of IFT88 depletion on IL-1 response signature over 24h. Bulk RNA profiles of control siRNA targetted (control) and IFT88 siRNA (IFT88) treated fibroblastic chondrocyte line, both treated with and without IL-1 beta in quiescent state.

研究目的：针对白细胞介素-1（interleukin-1, IL-1）下游的全基因组范围开展RNA测序（RNA sequencing），以探究IFT88基因敲减的作用范围与本质。
实验方法：将成纤维样软骨细胞分为四组：对照小干扰RNA（small interfering RNA, siRNA）文库处理组、靶向IFT88的siRNA文库处理组，每组均分别添加或不添加10ng/ml的白细胞介素-1β（interleukin-1β, IL-1β）。于0、1、2、4、8及24小时各收集三份重复样本的RNA。采用Illumina的TruSeq建库流程构建PolyA富集筛选的测序文库，通过Illumina HiSeq 4000平台进行75bp双端测序，每个样本的平均测序深度达2850万读对。使用Hisat2工具将测序读段比对至小鼠基因组。
实验结果：采用时间序列分析工具ImpulseDE2确定仅对照组的IL-1应答时间进程，并将该结果与实验组-对照组比较分析结果取交集。使用DESeq2工具开展IFT88处理组与对照组的逐点差异表达分析。
研究结论：本研究证实了24小时内IFT88基因敲减对IL-1应答特征的调控作用。本数据集包含经对照siRNA（对照组）与IFT88 siRNA（IFT88组）处理的成纤维样软骨细胞系的批量RNA表达谱，两组均分别设置IL-1β处理与未处理的静息状态样本。