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Next Generation Sequencing Facilitates Quantitative Analysis of CP5322-treated and Non-treated Transcriptomes in Kasumi-1 cells

ABSTRACT: Purpose: The goals of this study are designed to study the alteration of transcriptome profiling (RNA-seq) in CP5322-treated and non-treated cells. The drug was a herbal small molecular, which was designed as a HDAC inhibitor, called CP5322. TK1 was the group of CP5322-treated, and CK1 was the group of control. Methods: The mRNA profiles of drug-treated and non-treated cells were generated by next genenration sequencing, using Illumina platform. The clustering of the index-coded samples was performed on a cBot cluster generation system using HiSeq PE Cluster Kit v4-cBot-HS (Illumina) according to the manufacturer's instructions. After cluster generation, the libraries were sequenced on an Illumina platform and 150 bp paired-end reads were generated, which was called Raw Reads. And the low quality sequences and adaptor contamination were removed.Data processing was completed to obtain high-quality sequences (Clean Reads), and all subsequent analyses were based on Clean Reads. Results: Using the data analysis workflow, we mapped about 46 million sequence reads per sample to the human genome (h19) and identified 23 thousand transcripts in the drug-treated and non-treated cells. And the mapping rate was about 97%. RNA-seq data showed a significant alteration when the Kasumi-1 cells were treated by drug. Conclusions: The RNA-Seq data provided us a priori evidence for further research.

ORGANISM(S): Homo sapiens

PROVIDER: GSE142561 | GEO | 2020/03/01

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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Next Generation Sequencing Facilitates Quantitative Analysis of CP5322-treated and Non-treated Transcriptomes in Kasumi-1 cells

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