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RNA sequencing of isolated lung endothelial cells from Wildtype (WT) and PRR(Atp6ap2) endothelial cell-specific mutants

ABSTRACT: Purpose: ATP6AP2 is a single transmembrane protein that has been implicated in regulating various biological process including vascular angiogenesis. However, the exact role of ATP6AP2 during blood vessel development remains largely undefined. Here, we use an inducible endothelial cell (EC)-specific mouse to investigate the role of murine Atp6ap2 gene during both physiological and pathological angiogenesis of the postnatal retina, a well-established model system of angiogenesis. In this study we aimed to Identify what genes are differentially expressed upon loss of PRR(Atp6ap2) in ECs. Methods: ECs were isolated from lungs of both WT and PRR mutant mice at postnatal day 7 (P7). Total RNA was extracted from isolated lung ECs and quantified using Qubit RNA High Sensitivity Assay Kit. RNA integrity was determined using the Bioanalyzer RNA 6000 Nano assay kit. RNA library construction was performed with the TruSeq RNA Library Prep Kit v2 from Illumina. The resulting mRNA library was quantified using Qubit dsDNA High Sensitivity Assay Kit and verified using the Bioanalyzer DNA1000 assay kit. Verified samples were sequenced using the NextSeq 500/550 High Output Kit v2.5 (150 Cycles) on a Nextseq 550 system. Sequenced reads were aligned to the mouse (mm10) reference genome with RNA-seq alignment tool (version 2.0.1). The aligned reads were used to quantify mRNA expression and determine differentially expressed genes using the RNA-seq Differential Expression tool (version 1.0.1). Both alignment and differential expression analysis were performed using the tools in the BaseSpace Sequence Hub. Results: We found 1876 differentially expressed genes of which 823 genes were upregulated while 1053 genes were downregulated. Downregulated genes were enriched in biological processes such as angiogenesis, cell migration and extracellular matrix remodelling, while upregulated genes were enriched in DNA and cell cycle regulation. Conclusions: We identify PRR (ATP6AP2) to play an important role in regulation of the angiogenesis process.

ORGANISM(S): Mus musculus

PROVIDER: GSE179431 | GEO | 2021/07/07

REPOSITORIES: GEO

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Project description:Purpose: Zinc Finger MIZ-Type Containing 1 (Zmiz1) is a member of the PIAS family of protein and function as a transcriptional coactivator of Notch, Androgen Receptor (AR), p53, Estrogen Receptor (ER), and Smad3/4 . Despite Zmiz1 critical role in angiogenesis, its role in lymphatic vasculature is unknown. Here, we use HDLECs cell line to profile transcriptional changes upon Zmiz1 knockdown using siRNA. Methods: Total RNA was extracted from control and Zmiz1 siRNA transfected HDLECs using GeneJET RNA Purification Kit (Thermo Fisher Scientific, K0732) following the manufacturer instructions. RNA concentration and RNA integrity (RIN) number were determined using Qubit RNA High Sensitivity Assay Kit (Thermo Fisher Scientific, Q32852) and Bioanalyzer RNA 6000 Nano assay kit (Agilent, 5067-1511) respectively. RNA library was prepared using TruSeq RNA Library Prep Kit v2 (Illumina, RS-122-2001) according to the manufacturer instructions. mRNA library was quantified using Qubit dsDNA High Sensitivity Assay Kit (Thermo Fisher Scientific, Q32851) and verified using the Bioanalyzer DNA1000 assay kit (Agilent, 5067-1505). Verified samples were sequenced using the NextSeq1000/2000 P2 Reagents (200 Cycles) v3 (Illumina, 20046812) on a Nextseq1000/2000. Sequenced reads were aligned to the human (hg19) reference genome with RNA-Seq alignment tool (STAR aligner). The aligned reads were used to quantify mRNA expression and determine differentially expressed genes using the RNA-Seq Differential Expression tool (version 1.0.1). Both alignment and differential expression analysis were performed using the tools in the illumina BaseSpace Sequence Hub. Results: We found 2,228 differentially expressed genes of which 1,115 genes were upregulated while 1,113 genes were downregulated. Downregulated genes were enriched in biological processes such as lymph vessel development, cell migration and heart valve development. Conclusions: We identify Zmiz1 regulates Prox1 in lymphatic endothelial cells

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RNA sequencing of isolated lung endothelial cells from Wildtype (WT) and PRR(Atp6ap2) endothelial cell-specific mutants

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