Project description:Typical multiomics studies employ separate methods for DNA, RNA, and protein sample preparation, which is labor intensive, costly, and prone to sampling bias. We describe here a method for preparing high-quality DNA, RNA, and protein or peptides for whole genome sequencing, RNA sequencing, and whole proteome analysis from a single sample. This method utilizes a reversible protein tagging scheme to covalently link all proteins in a lysate to a bead-based matrix and nucleic acid precipitation and selective solubilization to yield separate pools of protein and nucleic acids. The proteins are then digested to generate peptides ready for mass spectrometric proteome analysis. We demonstrate the utility of this method to compare the genomes, transcriptomes, and proteomes of four triple-negative breast cancer cell lines with different degrees of malignancy. These data show the involvement of both RNA and associated proteins, and protein-only dependent pathways that distinguish these cell lines. We also demonstrate the utility of this multiomics workflow for tissue analysis using mouse brain, liver and lung tissue.
Project description:Intervention type:DRUG. Intervention1:Huaier, Dose form:GRANULES, Route of administration:ORAL, intended dose regimen:20 to 60/day by either bulk or split for 3 months to extended term if necessary. Control intervention1:None.
Primary outcome(s): For mRNA libraries, focus on mRNA studies. Data analysis includes sequencing data processing and basic sequencing data quality control, prediction of new transcripts, differential expression analysis of genes. Gene Ontology (GO) and the KEGG pathway database are used for annotation and enrichment analysis of up-regulated genes and down-regulated genes.
For small RNA libraries, data analysis includes sequencing data process and sequencing data process QC, small RNA distribution across the genome, rRNA, tRNA, alignment with snRNA and snoRNA, construction of known miRNA expression pattern, prediction New miRNA and Study of their secondary structure Based on the expression pattern of miRNA, we perform not only GO / KEGG annotation and enrichment, but also different expression analysis.. Timepoint:RNA sequencing of 240 blood samples of 80 cases and its analysis, scheduled from June 30, 2022..
Project description:Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways and the function of all RNAs in a certain functional state of a specific cell was studied, mainly including non-coding RNAs.The aim of this study was to compare lung tissue transcriptome analysis (RNA-SEQ) with microarray and quantitative reverse transcription polymerase chain reaction (QRT-PCR) methods for LPS-induced acute lung injury and to evaluate the optimal high-throughput data analysis protocol. Methods: LncRNA profiles of normal lung tissue and LPS-induced acute lung injury after 24h in mice were generated by deep sequencing, in triplicate, using Illumina HiSeq 4000 Results: A total of 8,610 lncRNAs were identified in the normal and LPS groups. Conclusions: Our study represents detailed analysis of lung tissue transcriptomes, withh biological replicates, generated by RNA-seq technology. Novel ideas are presented to expand our knowledge on the regulation mechanisms of lncRNA-related ceRNAs in the pathogenesis of ALI.
Project description:Idiopathic pulmonary fibrosis (IPF) is the prototypic progressive fibrotic lung disease with a median survival of 2-4 years. Injury to and/or dysfunction of alveolar epithelium are strongly implicated in IPF disease initiation, but what factors determine why fibrosis progresses rather than normal tissue repair occurs remain poorly understood. We previously demonstrated that ZEB1-mediated epithelial-mesenchymal transition (EMT) in human alveolar epithelial type II (ATII) cells augments TGF-β-induced profibrogenic responses in underlying lung fibroblasts by paracrine signalling. Here we investigated bi-directional epithelial-mesenchymal crosstalk and its potential to drive fibrosis progression. RNA sequencing (RNA-seq) of lung fibroblasts exposed to conditioned media from ATII cells undergoing RAS-induced EMT identified many differentially expressed genes including those involved in cell migration and extracellular matrix (ECM) regulation. We confirmed that paracrine signalling between AS-activated ATII cells and fibroblasts augmented fibroblast recruitment and demonstrated that this involved a ZEB1-tissue plasminogen activator (tPA) axis. In a reciprocal fashion, paracrine signalling from TGF-β-activated lung fibroblasts or IPF fibroblasts induced RAS activation in ATII cells, at least partially via the secreted protein, SPARC. Together these data identify that aberrant bi-directional epithelial-mesenchymal crosstalk in IPF drives a chronic feedback loop that maintains a wound-healing phenotype and provides self-sustaining pro-fibrotic signals.