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Next Generation Sequencing Facilitates Quantitative Analysis of MicroRNA-148a Inhibited Transcriptomes

ABSTRACT: Purpose: MicroRNA-148a regulates the differentiation of dendritic cells. The goals of this study are to identify genes and pathways invovled in the regulation. Methods: Monocytes isolated from wild type or microRNA-148a-/- BM were cultured with GM-CSF and IL-4, or not. Cells were collected at day 0 and day 3. Then, the next-generation libraries of mRNA were prepared using VAHTS mRNA-seq v2 Library Prep Kit for Illumina® (Vazyme, Nanjing, China). The Library quality was determined by Bioanalyzer 4200 (Agilent, Santa Clara, CA, USA). Then the mRNA-seq libraries were sequenced in HiSeq ⅹ10 system (Illumina, San Diego, CA, USA) on a 150bp paired-end run. The differentially expressed genes were selected as having more than 0.5 fold difference in their geometrical mean expression between the compared groups and a statistically significant p-value (<0.05) by analysis of DEseq2. The GO analysis on differentially expressed genes was performed with an R package: Clusterprofiler using a p<0.05 to define statistically enriched GO categories. Pathway analysis was used to determine the significant pathway of the differential genes according to Kyoto Encyclopedia of Genes and Genomes Database (http://www.genome.jp/kegg/) and DAVID Bioinformatics Resources 6.8 (https://david.ncifcrf.gov/). Results: Gene ontology analysis showed that obviously changed genes enriched in autoimmune disease related pathways, suggesting miR-148a is important in autoimmune responses. 990 protein-coding genes upregulated in their mRNA abundance in miR-148a-/- monocytes on day 0, and 1036 genes in miR-148a-/- monocytes on day 3. Among them, 136 genes were found upregulation on both day 0 and 3 in miR-148a-/- monocytes.

ORGANISM(S): Mus musculus

PROVIDER: GSE142511 | GEO | 2019/12/23

REPOSITORIES: GEO

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Next Generation Sequencing Facilitates Quantitative Analysis of Changed MicroRNAs in Psoriasis Patients.

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RNA-Seq analysis of CD115- monocytes and CD115+ monocytes from naïve and tumor-bearing mice

Project description:Purpose: The goals of this study are to compare CD115- monocytes to CD115+ monocytes from naïve and tumor-bearing mice (EL4) with high-throughput data analysis. Methods: CD115- monocytes and CD115+ monocytes were sorted by flow cytomentry separatly. Total RNA was extracted from cells using the RNeasy Mini Kit (QIAGEN, Hilden, Germany) and residual DNA was removed with a DNA-free Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. Samples were loaded onto a 1% agarose gel before electrophoresis. DNA-free conditions were confirmed by SYBR Gold staining (Invitrogen). The quantity and the quality of RNA was evaluated by Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific) and bioanalyzer (Agilent 2100; Agilent Technologies, Santa Clara, CA, USA). RNA-Seq libraries were prepared with 1 μg of total RNA from two biological replicates of each group using TruSeq Stranded mRNA LT Sample Prep Kit and analyzed by Illumina Sequencer (both from Illumina, San Diego, CA, USA). Results: We found 1,723 differetially expressed genes (DEGs) between CD115- monocytes and CD115+ monocytes (cutoff value was set at 2-fold). DEGs of interest are invoved in granulocyte, transcription factors, mononuclear phagocytes, and angiogenesis. Conclusions: Our study represents the first detailed DEGs analysis of CD115- monocytes and CD115+ monocytes from naïve and tumor-bearing mice. Our results show that CD115- monocytes may have a closer relationship with neutrophils or PMN-MDSCs than the CD115+ monocytes. Also, our overall transcriptomic analysis further suggests that CD115- M-MDSCs and monocytes are separate subsets of monocytic cells.

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MicroRNA-148a-3p in pericyte-derived extracellular vesicles improves erectile function in diabetic mice by promoting cavernous neurovascular regeneration

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2023-12-19 | GSE195533 | GEO

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