Project description:We developed SLIC-CAGE (Super-Low Input Carrier-CAGE) approach to capture 5'end of RNA polymerase II transcripts from as little as 5-10 ng of total RNA. The dramatic increase in sensitivity compared to existing CAGE methods is achieved by specially designed, selectively degradable carrier RNA. We tested SLIC-CAGE on Saccharomyces cerevisiae (BY4741 strain) and produced libraries from 1-100 ng of total cellular RNA. We also produced S. cerevisiae nAnT-iCAGE libraries as the current gold-standard CAGE libraries using the recommended 5 micrograms of total cellular RNA to assess the quality of SLIC-CAGE libraries produces with up to 1000-fold less material. We provide a direct comparison between SLIC-CAGE and the latest nanoCAGE protocol (libraries created using S. cerevisiae total RNA) and show that SLIC-CAGE produces unbiased libraries of higher complexity and quality than nanoCAGE. Finally, we provide SLIC-CAGE libraries on mouse embryonic stem cells (E14) using 5-100 ng of total cellular RNA as starting material.
Project description:An increasing number of non-coding RNAs (ncRNAs) are implicated in various human diseases including cancer; however ncRNA transcriptome of hepatocellular carcinoma (HCC) remains largely unexplored. We use CAGE (Cap Analysis of Gene Expression) to comprehensively map transcription start sites (TSSs) across different etiologies of human HCC as well as mouse HCC, with particular emphasis on ncRNAs distant from protein-coding genes. We find thousands of significantly up-regulated distal ncRNAs in HCC tumors compared to their matched non-tumors, which are as many as protein-coding genes. Moreover, we identify many LTR retroviral promoters activated in HCC tissues and expressed in a subfamily-specific manner, which account for approximately 20% of the up-regulated distal ncRNAs. The transcripts derived from LTRs, determined by 3' RACE, are multi-exon nuclear ncRNAs typically 0.5-2kb in length. This study sheds light on ncRNA transcriptome of human and mouse HCC. Expression profiles using CAGE for 37 mouse HCC. The human data are archived at dbGaP (phs000885.v1.p1). An umbrella BioProject has been created to associate the GEO and dbGaP BioProjects: PRJNA278792
Project description:We developed SLIC-CAGE (Super-Low Input Carrier-CAGE) approach to capture 5'end of RNA polymerase II transcripts from as little as 5-10 ng of total RNA. The dramatic increase in sensitivity compared to existing CAGE methods is achieved by specially designed, selectively degradable carrier RNA. We apply SLIC-CAGE on mouse primordial germ cells embryonic day (E) 11.5 - 2 biological replicates.
Project description:The CAGE experiment was performed in four different chemostat conditions to assess if and how much the Transcription start site landscape changes in the industrial relevant S. Cerevisiae strain CEN.PK113-7D in different conditions. CAGE was also used to obtain accurate TSS annotations for each expressed gene.
Project description:We used nAnT-iCAGE – unbiased single-nucleotide resolution method for genome-wide transcription start site (TSS) capture, to produce libraries from Saccharomyces cerevisiae total RNA. Our goal was to investigate S. cerevisiae core-promoters and assess the rules of transcription initiation in BY4741 strain grown in YPD media.
Project description:We hypothesized that mapping genetic variants associated with promoter and enhancer functions can provide novel insights into the mechanism through which eQTLs (expression quantitative trait loci) exert their effects on gene expression. To this end, we quantified genome-wide promoter usage and enhancer activity and tested the resulting molecular phenotypes for association with nearby genetic variants to discover cis-QTLs. To perform such analyzes, we have produced deep transcriptome profiling of 154 unrelated central European individuals, applying deepCAGE (Cap Analysis of Gene Expression) to nuclear enriched total RNA extracted from EBV transformed lymphoblastoid cell lines (LCLs). The LCLs were either purchased from Coriell Cell Repository (CEU, n=86) or from the GenCord collection (n=68, Gutierrez-Arcelus M et al. Elife 2013).
Project description:A comprehensive Chinese hamster ovary (CHO)-cell specific spectral library, containing extensive information of high-quality spectral ions covering more than 10k CHO cell proteins, was constructed to provide confident and reproducible protein identification and quantification in data-independent SWATH-MS analysis. The applicability of CHO spectral library was tested in the analyses of different CHO samples including whole cell lysate, harvested cell culture fluids, and downstream processing samples. The portability of CHO spectral library was also demonstrated in the processing and analyses of SWATH-MS data sets collected from multiple LC-MS instrumental setups and various CHO cell lines.
Project description:We detect the small RNAs subcellular distribution in breast cancer cell lines MCF-7 and MDA-MB-231, and normal cell line MCF-10A. Each cell line, we detected the nuclear and cytoplasmic small RNAs expression intensity; and then we could get the nuclear-cytoplasmic-ratio.
Project description:Here, we aimed to study metastasis mechanisms using spatial proteomics applied to the KM12 cell model of metastasis. KM12 cells were metabolically labelled using SILAC. SILAC has been successfully used for the analysis of proteome turnover and for the identification of changes in proteome localization as a consequence of DNA damage.10,23-24 Subcellular fractionation of the KM12 cells into five subcellular fractions corresponding to cytoplasm (CEB), plasma, mitochondria and ER/golgi membranes (MEB), nuclear (NEB), chromatin-bound (NEB-CBP) and cytoskeletal proteins (PEB) contributed to clarify the molecular mechanisms underlying CRC metastasis. Protein abundance and localization were measured in parallel for the five separate subcellular fractions, providing a map draft of the spatially deregulated protein complexes and networks in CRC metastasis.