strand specific sequencing of RNAs from MAoECs to determine the endothelial-specific expression profile of protein-coding and long non-coding RNAs Overall design: Total RNA was isolated from cultured MAoECs (passage 4) and processed for a strand-specific RNA sequencing. The RNA purity and integrity were assessed using the Fragment Analyzer Automated CE System (Advanced Analytical). A RQN of 8.8 and a 28S/18S ratio of 2.2 were considered acceptable for next generation sequencing assay. Five µg of DNase-treated RNA were used to prepare Massive Analysis of cDNA ends (MACE) libraries needed to perform a DNA-Methylation-Sequencing (Meth-Seq) PCR bias free quantification with TrueQuant Technology, followed by a high-throughput sequencing on the Illumina Genome Analyzer II system (GenXPro GmbH, Frankfurt, Germany). The procedure consist in the extraction of poly-adenylated RNA from 5 µg RNA and reverse transcribed with biotinylated poly(T) primers. cDNA is fragmented to an average size of 250 bp. Biotinylated ends are captured by streptavidin beads and ligated to modified adapters (TrueQuant DNA adapter, GenXPro). The libraries are amplified by PCR, purified by SPRI beads and sequenced (2 x 100 bp Illumina HiSeq2000 TrueSeq, 2 x 20 Mio. Reads poly-A selected paired-end reads). Paired end sequencing of both DNA strands from each end is required for fragment strand specificity.
miR-103 promotes endothelial maladaptation by targeting lncWDR59.
Specimen part, Subject
View SamplesGene expression profiling for identification of genes regulated by DNA methylation
Genome-wide screening of genes regulated by DNA methylation in colon cancer development.
Specimen part, Cell line
View Sampleswhole-transcriptom analysis of HT-29 and SW480 cells by HTA 2.0 microarray
S-Adenosylmethionine Treatment of Colorectal Cancer Cell Lines Alters DNA Methylation, DNA Repair and Tumor Progression-Related Gene Expression.
Specimen part, Cell line
View SamplesHigher order chromosome structure and nuclear architecture can have profound effects on gene regulation. We analyzed how compartmentalizing the genome by tethering heterochromatic regions to the nuclear lamina affects dosage compensation in the nematode C. elegans. In this organism, the dosage compensation complex (DCC) binds both X chromosomes of hermaphrodites to repress transcription two-fold, thus balancing gene expression between XX hermaphrodites and XO males. X chromosome structure is disrupted by mutations in DCC subunits. Using X chromosome paint fluorescence microscopy, we found that X chromosome structure and subnuclear localization are also disrupted when the mechanisms that anchor heterochromatin to the nuclear lamina are defective. Strikingly, the heterochromatic left end of the X chromosome is less affected than the gene-rich middle region, which lacks heterochromatic anchors. These changes in X chromosome structure and subnuclear localization are accompanied by small, but significant levels of derepression of X-linked genes as measured by RNA-seq, without any observable defects in DCC localization and DCC-mediated changes in histone modifications. We propose a model in which heterochromatic tethers on the left arm of the X cooperate with the DCC to compact and peripherally relocate the X chromosomes, contributing to gene repression. Overall design: RNA-seq profiles of C. elegans L1 wild type hermaphrodites, cec-4, met-2 set-25, and DPY-27 RNAi. RNA-seq profiles or C. elegans. Strains are N2 Bristol strain (wild type), RB2301 cec-4(ok3124) IV, and EKM99 met-2(n4256) set-25(n5021) III. Biological replicates for each strain/stage listed separately.
Anchoring of Heterochromatin to the Nuclear Lamina Reinforces Dosage Compensation-Mediated Gene Repression.
Specimen part, Subject
View SamplesDespite the importance of inter-cellular communication networks in regulating stem cell fate decisions, very little is known about the topology, dynamics, or functional significance. Using human blood stem cell cultures as an experimental paradigm, we present a novel bioinformatic approach to integrate genome-scale molecular profiles (transcriptome and secretome) and publicly available databases to reconstruct soluble factor-mediated inter-cellular signalling networks regulating blood stem cell fate decisions.
Dynamic interaction networks in a hierarchically organized tissue.
Specimen part
View SamplesRationale: Chronic Obstructive Pulmonary Disease (COPD) is considered a chronic inflammatory disease characterized by progressive airflow limitation and also has significant extrapulmonary (systemic) effects that lead to comorbid conditions. Very little is known about the pathomechanism of the disease.
Chronic obstructive pulmonary disease-specific gene expression signatures of alveolar macrophages as well as peripheral blood monocytes overlap and correlate with lung function.
Specimen part, Disease
View Samples