RNA-seq of In vitro differentiation of ciliated cells in ALI-cultured human airway epithelium
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ABSTRACT: The aim of this study was to analyze gene expression of primary airway epithelial cells during their differentiation in air-liquid interface ALI culture. Cells from one healthy donor were collected at different days of ALI culture (0,6,9,14 and 21). All samples were sequenced on Illumina NovaSeq6000 platform, in 2x150 mode, with a coverage of 100M reads/sample. Obtained data was later correlated with expression of 14 genes known to be related to airway epithelium differentiation obtained using qRT-PCR technique (TaqMan low density array, TLDA cards).
Project description:The aim of this study was to investigate the transcriptome of 54 primary samples of pediatric patients diagnosed with T-cell acute lymphoblastic leukemia. Samples were collected before treatment. Sequencing libraries were obtained with Illumina TruSeq Stranded mRNA protocol, with modified conditions of RNA fragmentation (90°C for 2 minutes). All libraries were sequenced on Illumina NovaSeq6000 platform, in 2x150PE mode (paired end sequencing with 150 nt reads), with a coverage of 150M reads/sample.
Project description:The aim of this study was to investigate the effect of hsa-miR-143-3p on global gene expression in T-cell acute lymphoblastic leukemia (T-ALL) and to unravel its direct target genes. For this purpose we selected Jurkat T-ALL cell line characterized by low expression of this miRNA. The cells were transduced either with pCDH-CMV-MCS-EF1_GFP_puro empty vector (System Biosciences) or pCDH-CMV-MCS-EF1_GFP pre-mir-143 vector, each variant in three biological replicates. Total RNA was isolated from each samples. Additionally, RISC complexes containing miRNAs bound to their target mRNAs were isolated by AGO2 immunoprecipitation and the obtained fraction of RISC-bound RNA was isolated from each sample. All 12 libraries (6 total RNA samples and 6 AGO2-RIP samples) were sequenced on Illumina NovaSeq6000 platform, in 2x150 mode, with a coverage of 60M paired reads/sample.
Project description:The aim of this study was to compare two different library preparation protocols: Illumina TruSeq Stranded mRNA (referred to as PA) and TruSeq Stranded Total RNA with Ribo-Zero Human/Mouse/Rat (RD) each with two different variants of the fragmentation step: standard fragmentation conditions (94°C for 8 minutes; further described as P1) and modified conditions (90°C for 2 minutes; further described as P2). The modification was expected to produce longer fragments. Each of the 5 tested T-ALL samples was sequenced using a combination of 4 different protocols: PA_P1, RD_P1, PA_P2, RD_P2 (for PA_P2 we performed two technical replicates). All 25 libraries were sequenced on Illumina NovaSeq6000 platform, in 2x150 mode, with a coverage of 150M reads/sample. High number of reads obtained for each of the samples allowed us to carry out a comprehensive comparison using various analysis methods aimed at identifying differentially expressed genes, alternative splicing events, gene fusions, somatic mutations and indels.
Project description:The aim of this study was to investigate the effect of hsa-miR-363-3p on global gene expression in T-cell acute lymphoblastic leukemia (T-ALL). For this purpose we selected DND-41 T-ALL cell line characterized by high expression of this miRNA. The cells were transduced either with miRZip pGreenPuro scrambled vector (System Biosciences) or miRZip anti-hsa-miR-363-3p vector, each variant in three biological replicates. All 6 libraries were sequenced on Illumina NovaSeq6000 platform, in 2x150 mode, with a coverage of 60M paired reads/sample.
Project description:We identified inactivating mutations in NEK10, a poorly characterized human protein kinase, in a novel human bronchiectasis syndrome. In order to understand effects of loss of its function on the airway phosphoproteome, NEK10 was CRISPR/Cas9 targeted in human airway air-liquid interface (ALI) cultures with 2 independent guides and assayed by iron-enrichment phosphoproteomics.
Project description:The regeneration of the airway mucociliary epithelium involves several sequential events including migration, proliferation, polarization and final differentiation (i.e ciliogenesis). The airway mucociliary epithelium is consituted of three main cell types : ciliated cells, secretory cells and basal cells. We used microRNA microrrays to investigate the signature of microRNA during the four step of regeneration of the airway epithelium. Four time points (ALI-D0, ALI-D7, ALI-D14, ALI-D21) of regeneration of the airway epithelium for 3 donors.
Project description:We want to observe the dynamics of MTEC differentiation by looking at the gene expression differences at two points: ALI (Air-Liquid Interface) 4 days, and ALI 7 days.
Project description:Normal human bronchial epithelial (NHBE) cells cultured in an air-liquid interface (ALI) system form a polarized, pseudostratified epithelium composed of basal, ciliated and goblet cells that closely resemble the in vivo airway epithelium structure. ALI cultures of NHBE cells provide a unique in vitro system to investigate airway epithelial biology, including developmental, structural and physiologic aspects. In this study, we wanted to investigate mRNA expression patterns during airway epithelium differentiation. By using microarrays, we studied the changes in expression of mRNAs in normal human bronchial epithelial cells as they differentiate from an undifferentiated monolayer to a differentiated pseudostratified epithelium after 28 days of air-liquid interface (ALI) culture, when epithelial cells differentially express basal, ciliated and goblet cell markers. Normal human bronchial epithelial cells were cultured in an air-liquid interface (ALI) system and harvested at three different time-points: subconfluent, confluent and day 28 of ALI. Samples were processed for total RNA extraction and hybridization on Affymetrix microarrays. All the experiments were performed by triplicate.
Project description:The regeneration of the airway mucociliary epithelium involves several sequential events including migration, proliferation, polarization and final differentiation (i.e ciliogenesis). The airway mucociliary epithelium is consituted of three main cell types : ciliated cells, secretory cells and basal cells. We used microRNA microrrays to investigate the signature of microRNA during the four step of regeneration of the airway epithelium. Four time points (ALI-D0, ALI-D7, ALI-D14, ALI-D21) of regeneration of the airway epithelium for 3 donors.
Project description:Normal human bronchial epithelial (NHBE) cells cultured in an air-liquid interface (ALI) system form a polarized, pseudostratified epithelium composed of basal, ciliated and goblet cells that closely resemble the in vivo airway epithelium structure. ALI cultures of NHBE cells provide a unique in vitro system to investigate airway epithelial biology, including developmental, structural and physiologic aspects. MicroRNAs (miRNAs) are short, single-stranded, non-coding RNAs of 20-23 nucleotides that down-regulate gene expression by either inducing degradation of target mRNAs or impairing their translation. They are phylogenetically well conserved, which probably implies an important role of miRNAs in biological processes. In this way, we wanted to shed some light on miRNA specific roles and the relationship with their mRNA targets during airway epithelium differentiation. By using microarrays, we studied the changes in expression of microRNAs in normal human bronchial epithelial cells as they differentiate from an undifferentiated monolayer to a differentiated pseudostratified epithelium after 28 days of air-liquid interface (ALI) culture, when epithelial cells differentially express basal, ciliated and goblet cell markers. Normal human bronchial epithelial cells were cultured in an air-liquid interface (ALI) system and harvested at three different time-points: subconfluent, confluent and day 28 of ALI. Samples were processed for total RNA (including small RNAs) extraction and hybridization on Affymetrix microarrays. All the experiments were performed by triplicate.