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

ABSTRACT: 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.

ORGANISM(S): Mus musculus

PROVIDER: GSE128532 | GEO | 2020/07/02

REPOSITORIES: GEO

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Project description:To determine IL-17-induced global transcriptome changes in midbrain neurons derived from induced pluripotent stem cells (iPSC) from three sporadic Parkinson's disease (PD) patients and three age- and sex-machted controls, deep RNA sequencing (RNA-Seq) of IL-17-treated and untreated PD and control iPSC-dderived midbrain neurons was performed. Total RNA was isolated from untreated and IL-17-treated cells with the TruSeq RNA Sample Preparation Kit v2 (Illumina). RNA libraries were quantified using the KAPA SYBR FAST ABI Prism Library Quantification Kit (Kapa Biosystems) and cluster generation was performed on the cBot with the TruSeq SR Cluster Kit v3 (Illumina). The sequencing run was performed on a HiSeq 1000 instrument (Illumina) using the indexed, 50 cycles single read (SR) protocol and the TruSeq SBS v3 Kit (Illumina). Image analysis and base calling resulted in .bcl files that were then converted into .fastQ files by the CASAVA1.8.2 software. FastQ files were aligned to the human genome (hg19) using STAR.and annotated with gencode.v19. DESeq2 was used to determine differential expression. Criteria to determine significantly dysregulated genes were as follows: adjusted p-value below 0.05 and log2FC (fold change) of greater than one. Only genes with a mean expression value of greater than one RPKM (reads per kilobase per million mapped reads) throughout the dataset were considered. Control and PD samples were analyzed as two independent datasets. Upon IL-17 treatment only 17 genes were found to be dysregulated in controls but 125 genes were dysregulated in iPSC-derived midbrain neurons from PD patients. The 125 IL-17-dependent genes in PD iPSC-derived neurons separated the treated from untreated PD samples using an unsupervised, hierarchical clustering applying an Euclidean distance metric. More detailed study information can be found in Sommer A, Maxreiter F, Krach F, Fadler T, Grosch J, Maroni M, Graef D, Eberhardt E, Riemenschneider MJ, Yeo GW, Kohl Z, Xiang W, Gage FH, Winkler J, Prots I, Winner B. Th17 Lymphocytes Induce Neuronal Cell Death in a Human iPSC-Based Model of Parkinson's Disease. Cell Stem Cell. 2018 Jul 5;23(1):123-131.e6. doi: 10.1016/j.stem.2018.06.015. PMID: 29979986

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

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