Project description:RNA-seq was performed on FACS-isolated stem cells from the mouse intestinal epithelium. The purpose of the experiment was to compare the transcriptomes of stem cells enriched for recently generated (young) mitochondria and stem cells enriched with old mitochondria. The dataset also includes samples of stem cells with young mitochondria deliberately contaminated with a small amount of Paneth cells, to control for accidental Paneth cell contamination in the sorting scheme.
Project description:Environmental stressors such as repeated social defeat may trigger powerful activation of sub-conscious parts of the brain. We examine the consequences of such stress in male rats on the pituitary gland. We prepared 20 Sprague Dawley rats. 10 of them were exposed to stress induced by the resident-intruder paradigm while the other 10 were controls. After dislocation of the neck under isoflurane anaesthesia, the pituitary gland was harvested. Total RNA was isolated from the tissues and used in mRNA sequencing.
Project description:Purpose: The goals of this study were to identify quantitative gene expression differences between wild type, Musashi1 null, Msuashi2 null and Musashi1/Musashi2 null MIAPaCa2 pancreatic cancer cells mRNA profiles of MIA PaCa-2 cancer cells were generated by deep sequencing, in triplicate, using Illumina HiSeq2500.
Project description:Background: hnRNP K is an RNA-binding protein that's been implicated in oncogenesis, particularly in hematological disorders. Intent: To determine the impact of hnRNP K overexpression on RNA expression in murine fetal liver cells. Experimental Workflow: Fetal liver cells were subjected to retrovial transduction with either empty vector GFP or hnRNP K GFP. Purified GFP+ cells were flow sorted, and RNA was extracted from the GFP+ population, upon which RNA sequencing was performed.
Project description:The histone variant H2A.B3 (H2A.Bbd/H2A.Lap1) has a tissue-specific expression pattern and is found to be most highly expressed during spermatogenesis. In order to investigate the role of H2A.B3 in chromatin packaging and regulation of spermatogenesis we created a line of TALEN-edited FVBN mice in which a deletion was introduced into the single exon of the H2AFB3 gene. The deletion was confirmed by Sanger sequencing and off-target effects investigated using exome sequencing. The deletion is visible in the RNA-Seq data of KO mouse, and the deleterious effect on the H2A.B3 protein has been confirmed using immunohistochemistry.
Project description:In mouse aorta endothelial cells, populations of endothelial vascular progenitor (EVP) and differentiated (D) cells could be identified by CD31 (lo/hi) and VEGFR2 (lo/hi) expression. These populations were FACS sorted and paired-end bulk RNA-sequencing was performed.
Project description:Affymetrix Human Gene 1.1 ST Array profiling of 285 primary medulloblastoma samples. Total RNA was extracted from primary medulloblastoma samples and hybridized to Affymetrix Human Gene 1.1 ST Arrays (24-Array Plates) according to the manufacturer's instructions.
Project description:Medulloblastoma is subdivided into different subgroups: WNt, SHH, Group 3 and Group 4. Since these subgroups are associated with different OS and metastasis rates it is crucial to understand them better. Six medulloblastoma cell lines, DAOY, ONS-76, D458, HD-MB03, CHLA-01-MED, CHLA-01R-MED, have been sequenced to compare them with medulloblastoma patient data. Methods: Medulloblastoma cell lines representating the different subgroups have been cultured and cell were harvested and RNA was isolated when 70% confluency was reached. In detail, DAOY and D458 were grown in DMEM (Thermo Fisher) with 10% FBS (HyClone, Thermo Fisher), ONS-76 and HD-MB03 were grown in RPMI 1640 (Sigma-Aldrich) with 10% FBS and CHLA-01-MED and CHLA-01R-MED were grown in DMEMF12 supplemented with B27, 20 ng/ml EGF and 20 ng/ml bFGF (all Thermo Fisher). All cells were maintained at 37 °C in a humidified atmosphere containing 5% CO2. During the course of this study, all cell lines were routinely confirmed to be mycoplasma negative (MycoAlert, Lonza, Basel, Switzerland).Cell pellets of at least 100,000 cells were washed with HBSS and frozen in liquid nitrogen. For homogenization, ceramic spheres (Lysing Matrix D, MP Biomedicals, Santa Ana, California, USA) and the FastPrep-24 homogenizer was used (MP Biomedicals, speed 4 m/s, tube holder MP:24*2 and time 20 s). Total RNA was isolated from 2D pellets using the NucleoSpin RNA Plus Kit (Macherey-Nagel, Düren, Germany) according to the manufacturer’s instructions. In total, 3 biological replicates of each cell line were processed respectively. RNA amount was determined using the Qubit RNA BR kit with the Qubit 4 (both ThermoFisher). Library preparation and RNA sequencing (transcriptome sequencing including lncRNA on Illumina PE150) were performed by Novogene (Cambridge, UK) Company Limited, Cambridge, UK. Samples with less than 100 ng or with non-qualifying RIN values were excluded from the sequencing. All prepared libraries successfully passed Novogene’s internal quality control checks and were sequenced. Following sequencing, quality control of the sequencing data was performed that confirmed all samples had high quality scores, indicating good technical performance of the sequencing. We used FastQC to perform quality checks of raw RNA data followed by adapter and low quality read filtering using the Cutadapt package (version 1.16.6) [reference]. The trimmed paired-end sequences were aligned with the human genome (hg38) and Gencode annotation (v35) using the STAR (version 2.7.5b) alignment tool. Unique reads from genomic alignment were processed and we used the featureCount tool for transcript abundance quantification. STAR read counts were used as input into edgeR. Genes with read counts greater than 10 in three or more samples were kept for subsequent analyses. After normalization analyses, counts per million (cpm) on a log2 scale were used for downstream exploratory analyses.
Project description:Our group is interested in epithelial-to-mesenchymal transition (EMT), in particular, TGF-beta induced EMT. TGF-beta signalling has been shown to be an important factor in the induction of EMT and it has been demonstrated that adding TGF-beta to epithelial cells in culture is a convenient way to study the process of EMT. âIn response to TGF-beta, Smad2 and 3 are activated, and form complexes with Smad4, which then regulate transcription of target genes through interactions with other DNA binding transcription factors. In the induction of EMT, the activated Smads mediate transcriptional regulation through three families of transcription factors, resulting in repression of epithelial marker gene expression and activation of mesenchymal gene expressionâ (Xu J, et al. 2009) <br></br> Also investigated in this study is the role of H2A.Z in EMT. H2A.Z is an evolutionary conserved and a metazoan essential histone variant of the H2A class. Mice deficient in H2A.Z die during early development but the reason for this is unknown (Faast et al. 2001). Previously, our laboratory showed that the loss of H2A.Z in Xenpous laevis impaired cell movement required for the formation of the mesoderm and neural crest (Ridgway et al. 2004). Given that mesoderm formation is critically dependent upon EMT, we therefore wondered whether H2A.Z might be a chromatin regulator of EMT. We transfected MDCK cells with a lentiviral vector to express a construct encoding an shRNA targeting canine H2A.Z as we wanted to test the hypothesis that H2A.Z is involved in the maintenance of cellular identity and that its loss might trigger de-differentiation. <br></br> In order to investigate changes in gene expression associated with TGF-beta induced epithelial-to-mesenchymal transition (EMT) we performed paired end RNA-Seq of poly-A selected mRNA in untreated and TGFb-treated MDCK cells. The MDCK cell line has been extensively used as a model system for EMT because they convert fully from the epithelial to the mesenchymal state in response to TGF-beta. Gene expression profiles were also generated from MDCK cells in which H2A.Z was knocked down using shRNA.<br></br>Please note that ChIP-seq data generated in conjunction to this RNA-seq data set were also deposited at ArrayExpress under accession number E-MTAB-5637 ( https://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-5637 ).
Project description:This experiment seeks to elucidate the functional role of MYB73 in Arabidopsis thaliana siliques via differential gene expression (DGE) analysis. Total RNA were extracted from pooled Arabidopsis siliques at 12 days after flowering (DAF) for 3 biological replicates from WT and MYB73-OE lines. RNA-seq libraries were generated using NEBNext Ultra II Directional RNA Library Prep Kit (New England Biolabs) for Illumina according to the manufacturer’s instruction and sequenced with Novaseq-6000 (Illumina) using paired-end sequencing with read lengths of 150 base pairs at sequencing depths of ~ 2 million reads per sample.