Project description:Human hepatocytes, HepaRG cells and HCC HepG2 cells were treated with 50nM of Microcystin-LR and cylindrospermopsin (CYN) to observe changes in gene expression.
Project description:We analyzed the effect of knockdown of miR-214 compared to wild type on the expression of genes and pathways involved in prostate cancer cells.
Project description:We chronically exposed HepaRG (differentiated hepatic cells that retain primary human hepatocytes characteristics) and HepaG2 (liver hepatocarcinoma cells) liver cells to physiologically relevant concentrations of Cadmiun and PFOAs chemicals (10nM) to observed how these affect global gene expression in these cellular models.
Project description:We investigated how selective mutation in STK11 or KEAP1 alters the transcriptional profile of cancer cells, and how the transcriptional profile of co-mutant cells might uniquely affect cancer-related pathways including control of proliferative potential, metabolic homeostasis, and cell death. We performed bulk RNAseq on none, single or double mutation of these genes in single cell knockout clones (n=3 per group) from both H358 and H292 cell lines.
Project description:We found that the fungus, Debaryomyces hansenii (D. hansenii), is enriched in inflamed intestinal tissue from patients of Crohn's disease and its administration to mice impairs colonic wound healing in multiple models of colonic injury and repair. To understand the mechanism, we isolated bone marrow derived macrophages from mice and stimulated them in vitro with a pure isolate of D. hansenii. Total RNA was isolated at multiple time points and assayed for transcriptomic analysis.
Project description:We generated C57BL/6 mice lacking Bmp10 and/or Bmp9 utilizing the Cre-loxP system. Briefly, Bmp9 constitutive deletion resulted from the replacement of exon 2 by a neomycin resistance cassette. Because Bmp10 deletion leads to early embryonic lethality, we used the tamoxifen-inducible Cre system to generate Bmp10-cKO mice (Rosa26-CreERT2;Bmp10lox/lox) by crossing Rosa26-CreERT2 mice with Bmp10lox/lox mice that possess loxP sites flanking exon 2. To generate double-KO (DKO) mice, we crossed these Rosa26-CreERT2;Bmp10lox/lox mice with Bmp9-KO mice. At the age of 8 weeks, mice were treated with tamoxifen (Sigma) by intraperitoneal injection once a day for 5 days at a dosage of 50 mg/kg. At the age of 5 months, Wild Type and DKO mouse lung tissue was flash frozen in liquid nitrogen and stored at -80°C. RNA extraction, RNA sample quality assessment, RNA library preparation, sequencing and raw data analysis were conducted at GENEWIZ, Inc. (South Plainfield, NJ, USA). Total RNA was extracted from frozen tissue using the Qiagen RNeasy Plus Mini kit. RNA samples were quantified using Qubit 2.0 Fluorometer (Life Technologies, Carlsbad, CA, USA) and RNA integrity was checked with Agilent TapeStation (Agilent Technologies, Palo Alto, CA, USA). rRNA depletion was performed using Ribozero rRNA Removal Kit (Illumina, San Diego, CA, USA). RNA sequencing library preparation used NEBNext Ultra RNA Library Prep Kit for Illumina by following the manufacturer’s recommendations (NEB, Ipswich, MA, USA). Briefly, enriched RNAs were fragmented for 15 minutes at 94 °C. First strand and second strand cDNA were subsequently synthesized. cDNA fragments were end repaired and adenylated at 3’ends, and universal adapter was ligated to cDNA fragments, followed by index addition and library enrichment with limited cycle PCR. Sequencing libraries were validated using the Agilent Tapestation 4200 (Agilent Technologies, Palo Alto, CA, USA), and quantified by using Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA) as well as by quantitative PCR (Applied Biosystems, Carlsbad, CA, USA). The sequencing libraries were clustered on one lane of a flowcell. After clustering, the flowcell was loaded on the Illumina HiSeq 4000 instrument (or equivalent) according to manufacturer’s instructions. The samples were sequenced using a 2x150 Paired End (PE) configuration. Image analysis and base calling were conducted by the HiSeq Control Software (HCS). Raw sequence data (.bcl files) generated from Illumina HiSeq was converted into fastq files and de-multiplexed using Illumina's bcl2fastq 2.17 software. One mis-match was allowed for index sequence identification. After investigating the quality of the raw data, sequence reads were trimmed to remove possible adapter sequences and nucleotides with poor quality using Trimmomatic v.0.36. The trimmed reads were mapped to the the Mus musculus GRCm38 reference genome available on ENSEMBL using the STAR aligner v.2.5.2b. Gene counts were calculated from uniquely mapped reads using feature Counts from the Subread package v.1.5.2. Only unique reads that fell within exon regions were counted. The gene hit counts table was then used for downstream differential expression analysis. A differential gene expression analysis between WT and DKO groups of samples was performed using the R-package DESeq2 (Wald test).
Project description:Genomic alterations that activate Fibroblast Growth Factor Receptor 2 (FGFR2) are common in intrahepatic cholangiocarcinoma (ICC), a deadly bile duct malignancy. FGFR kinase inhibitors (FGFRi) have shown promising efficacy against FGFR2+ ICC in clinical trials, leading to the regulatory approval of the ATP-competitive FGFR inhibitors, pemigatinib and infigratinib (BGJ398), for this subset of patients who failed standard treatment . However, the objective response rate (ORR) for each FGFR inhibitor (FGFRi) studied to date in FGFR2+ ICC is <45% and disease progression invariably arises within ~6-12 months. By employing high-throughput drug screens and signaling studies, we identified signaling feedback via the EGFR pathway as a major mediator of adaptive resistance to FGFR kinase inhibition in a set of patient-derived ICC models. To further gain insights into the synergistic effects of the EGFR/FGFR combination and address the mechanisms underlying the survival pathway reactivation, we performed RNA sequencing in our FGFR-driven ICC models (ICC21 and ICC10-6). For these studies, we treated FGFR2 fusion+ ICC cell lines ICC21/ICC10-6 with four conditions (DMSO/Infigratinib/Afatinib/Combo) for 4 hours followed by RNA sequencing.
Project description:The objective of this study was to decipher the metabolism expressed by Lactobacillus delbrueckii subsp. delbrueckii CIRM-BIA865 during soy juice fermentation using transcriptomics. The whole genome was sequenced, assembled and annotated. CIRM-BIA865 was then used to ferment soy juice to produce a soy-based yogurt. Samples were analysed in kinetics during fermentation, at pH values of 6.5, 6, 5 and 4.6. RNA from CIRM-BIA865 were extracted and sequenced using paired-end Illumina. Reads were mapped using Bowtie2 on previously obtained genome of CIRM-BIA865. No mismatch were allowed. Reads mapped on CDS were counted using htseqcount.List of differentially expressed (DE) genes between two successive sampling times (determined by pH) were generated using DEseq2 with a modified t-test and a p-value adjusted by Bonferoni inferior to 0.05. Fold changes expressed how many times genes were induced along the fermentations.
Project description:To identify if genes is regulated by time of day in human tendinopathic tendon, RNAseq analysis was performed on biopsies taken from tendinopathic and contralateral patella tendon 12 hours apart in young tendinopathic subjects (9 AM and 9 PM).
Project description:Stress-related illness represents a major burden on health and society. Understanding the molecular mechanisms underlying stress responses is an important step to understand these diseases, and design treatments. Sexual dimorphism in stress responses have been extensively reported, with depression and most anxiety disorders being more prevalent in women. Corticotrophs of the anterior pituitary play a central role in the generation of hormonal stress responses, by secreting the stress hormone ACTH in response to stressful stimuli. However, their role in contributing to sexually dimorphic responses of the HPA axis is very poorly understood. We performed bulk RNA-sequencing of FACS-sorted corticotrophs from male and female POMC-GFP mice to determine the molecular determinants of sexual dimorphic traits in the activity of these cells, revealing extensive differential gene expression at the transcriptional level. This includes more than 71 mRNAs encoding ion channel subunits, which could be involved in the generation of the sexual dimorphism in their electrical activity.