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Analyses of the transcriptional and alternative splicing changes in mdf-1 in comparison to WT via RNA sequencing

ABSTRACT: Purpose: Understand the transcriptional changes and alternative splicing defects associated to the pleiotropic defects observed in mdf-1 mutants. Methods: Total RNA was extracted from 3 different biological replicates containing 12-day-old seedlings of WT and mdf-1 using the innuPREP Plant RNA kit (Analytik Jena Bio solutions). RNA quality assessment, library preparation, sequencing and bioinformatic analyses were performed by Novogene Co. Integrity and quantitation of the extracted RNA were measured using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies, CA, USA). With 1 µg RNA per sample as input material, sequencing libraries were subsequently generated using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB) following the manufacturer’s recommendations. Index codes were also added to attribute sequences to each sample. Clustering of the index-coded samples was carried out on a cBot Cluster Generation System using PE Cluster Kit cBot-HS (Illumina) following manufacturer’s instructions. Afterwards, library preparations were sequenced on an Illumina NovaSeq 6000 platform and approximately 50 million 150 paired end reads per sample were generated. To ensure the high quality of the samples, only clean data was used for subsequent analyses. This was achieved after the removal of reads containing adapter and poly-N sequences and reads with low quality from the raw data. Mapping against the Arabidopsis TAIR10 reference genome was performed using the HISAT2 software. Reads Per Kilobase of exon model per Million mapped reads (RPKM) of each gene was calculated based on the length of the gene and reads count mapped to this gene. Differential expression analyses between mdf-1 and WT were carried out using the DESeq2 R package with the Benjamini and Hochberg’s approach for adjusting P values according to the False Discovery Rate (FDR). Genes with adjusted p-value < 0.05 were considered as differentially expressed. Alternative splicing (AS) analysis was performed with the rMATS software. Events with adjusted p-value < 0.05 were considered as alternatively spliced. Results: Loss of MDF function is associated with growth defects, impaired transition to mitosis, increased endoreduplication and spontaneous cell death in meristems. This was associated with strong upregulation of stress-induced genes and down-regulation of mitotic regulators and a subset of genes required for essential developmental processes. MDF is also required for the correct splicing of numerous transcripts including transcription factors and cell cycle associated genes.

ORGANISM(S): Arabidopsis thaliana

PROVIDER: GSE197898 | GEO | 2022/09/26

REPOSITORIES: GEO

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Project description:Purpose: To obtain an overview of the metabolic changes associated to the absence of KpsM in Synechocystis, in particular on carbon-related metabolic pathways Methods: The trancriptomes of Synechocystis wild type and kpsM mutant were analyzed by RNA sequencing, using three biological replicates. Cultures were grown until reaching an OD730nm of 1.5. Sequencing libraries were generated using NEBNext® Ultra TM RNA Library Prep Kit for Illumina®. PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system. The clustering of the index-coded samples was performed on a cBot Cluster Generation System using PE Cluster Kit cBot-HS (Illumina). Raw data (raw reads) of FASTQ format were firstly processed through in-house scripts. In this step, clean data (clean reads) were obtained by removing reads containing adapter and poly-N sequences and reads with low quality from raw data. Paired-end clean reads were mapped to the reference genome using HISAT2 software.HTSeq was used to count the read numbers mapped of each gene, including known and novel genes. And then RPKM of each gene was calculated based on the length of the gene and reads count mapped to this gene. RPKM, Reads Per Kilobase of exon model per Million mapped reads, considers the effect of sequencing depth and gene length for the reads count at the same time, and is currently the most commonly used method for estimating gene expression levels. Differential expression analysis between two conditions/groups (three biological replicates per condition) was performed using DESeq2 R package. DESeq2 provides statistical routines for determining differential expression in digital gene expression data using a model based on the negative binomial distribution. The resulting P values were adjusted using the Benjamini and Hochberg’s approach for controlling the False Discovery Rate (FDR). Genes with an adjusted P value < 0.05 found by DESeq2 were assigned as differentially expressed. Results: Approximately 700 genes (out of the 3636 identified and 215 quantified, representing a coverage of 85,12%) were significantly differentially expressed (P 216 value < 0.05) in the kpsM mutant compared to the wild type. Of those, 406 were down217 regulated, while 297 were up-regulated. Conclusions: Overall, our transcriptomic data indicates alterations in the mechanisms of energy production and conversion in the kpsM mutant compared to the wild type. The approach resulted in the identification of altered levels of transcripts belonging to a variety of functional categories and highlighting a number of key metabolic processes affected by the disruption of kpsM, namely photosynthesis, oxidative phosphorylation and carbon metabolism.

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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Analyses of the transcriptional and alternative splicing changes in mdf-1 in comparison to WT via RNA sequencing

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