Riboseq and Quantseq of neuroblastoma cell line SH-SY5Y expressing MAPT-AS1 constructs
Ontology highlight
ABSTRACT: The aim of this experiment was to assess the on- and off-target effects of MAPT-AS1 expression, and whether mutations/deletions to MAPT-AS1 alter these effects. SHSY5Y cells stably expressing variants of MAPT-AS1 were analyzed by Riboseq and Quantseq.
Project description:QuantSeq-Rev method to generate highly strand-specific next-generation sequencing (NGS) libraries enabling transcript quantification and identification of the 3'end of polyadenylated RNAs
Project description:The main aim was to determine how polyadenylation site usage is affected by osteoarthritis. Age matched tissue samples were obtained from both healthy individuals and those with late stage osteoarthritis, total RNA was isolated and then QuantSeq Reverse library produced using commercial kit (Lexogen) before sequencing
Project description:Tau (MAPT) is a microtubule-associated protein causing frequent neurodegenerative diseases or inherited frontotemporal lobar degenerations. Emerging evidence for non-canonical functions of Tau in DNA protection and P53 regulation suggests its involvement in cancer. Indeed, Tau expression correlates with cancer-specific survival or response to microtubule therapeutics. These data may imply common molecular pathways involved in the pathogenesis of neurodegenerative disorders and cancer. To bring new evidence that Tau represents a key protein in cancer, we present an in silico pan-cancer analysis of MAPT transcriptomic profile in over 11000 clinical samples and over 1300 pre-clinical samples provided by the TCGA and the DEPMAP datasets respectively. We completed this analysis by exploring a possible interplay of MAPT with wild-type or mutated P53. Then, we calculated the impact of MAPT expression on clinical outcome and drug response. Overall, the results support a relevant role of the MAPT gene in several cancer types, although the contribution of Tau to cancer appears to very much depend on the cellular context.
Project description:TGFB2-AS1 is a long non-coding RNA which is induced by ΤGFβ signaling. In order to assess the importance of TGFB2-AS1 on the regulation of gene expression, we performed an AmpliSeq transcriptomic array in human keratinocytes (HaCaT), which stably over-express TGFB2-AS1 or control pcDNA3 empty vector. In addition, cells were stimulated with TGFβ1 for 24 hours, in order to observe the effects of TGFB2-AS1 on gene expression, downstream of TGFβ signaling. RNA from the following four conditions was used in this experiment: 1) pcDNA3, 2) pcDNA3+TGFβ1, 3) pcDNA3-TGFB2-AS1, 4) pcDNA3-TGFB2-AS1+TGFβ1. Biological triplicates were used per condition.
Project description:To assess how LARP6 affects mRNA localisation to cell-protrusions, we transfected MDA-MB231 cells with either non-targeting control siRNA or LARP6 siRNA for 72 hrs, before fractionating the cells into protrusion and cell-bodies, and analysed total RNA from each fraction via Lexogen QUANTSEQ FWD 3' mRNA sequencing. Protrusion induction was done for 2 hrs on 3 micron transwell filters (Corning). The experiment was performed twice using two independent batches of cells, and sequencing was carried out on an Illumina NextSeq 500 sequencer.
Project description:QuantSeq-Rev method to generate highly strand-specific next-generation sequencing (NGS) libraries enabling transcript quantification and identification of the 3'end of polyadenylated RNAs
Project description:Objectives: Long non-coding RNAs (lncRNAs) have been shown to play important roles in the development and progression of cancer. However, functional lncRNAs and their downstream mechanisms are largely unknown in the molecular pathogenesis of esophageal adenocarcinoma (EAC) and its progression. Design: lncRNAs that are abnormally upregulated in EACs were identified by RNA-seq analysis, followed by quantitative RT-PCR (qRTPCR) validation using tissues from 31 EAC patients. Cell biological assays in combination with siRNA-mediated knockdown were performed in order to probe the functional relevance of these lncRNAs. Results: We discovered that a lncRNA, HNF1A-AS1, is markedly upregulated in human primary EACs relative to their corresponding normal esophageal tissues (mean fold change 7.2, p<0.01). We further discovered that HNF1A-AS1 knockdown significantly inhibited cell proliferation and anchorage independent growth, suppressed S-phase entry, and inhibited cell migration and invasion in multiple in vitro EAC models (p<0.05). A gene ontological analysis revealed that HNF1A-AS1 knockdown preferentially affected genes that are linked to assembly of chromatin and the nucleosome, a mechanism essential to cell cycle progression. The well-known cancer-related lncRNA, H19, was the gene most markedly inhibited by HNF1A-AS1 knockdown. Consistent to this finding, there was a significant positive correlation between HNF1A-AS1 and H19 expression in primary EACs (p<0.01). In order to identify novel oncogenic lncRNAs in esophageal adenocarcinogenesis, we carried out RNA-seq of a matched NE-BE-EAC tissue pair
Project description:H9 human ESCs (H9 line) were cultured in BMP4 cultured medium on Matrigel-coated plates. Colonies were passaged for maintanence by Gentle Cell Dissociation Reagent