Project description:miRNA array comparing the transcription profile of control rats and rats after intra-hippocampal pilocarpine-induced Status Epilepticus (PILO-SE).
Project description:Recent transcriptome analysis indicates that >90% of human genes undergoes alternative splicing, underscoring the contribution of differential RNA processing to diverse proteomes in higher eukaryotic cells. The polypyrimidine tract binding protein PTB is a well-characterized splicing repressor, but PTB knockdown causes both exon inclusion and skipping. Genome-wide mapping of PTB-RNA interactions and construction of a functional RNA map now revealed that dominant PTB binding near a competing constitutive splice site generally induces exon inclusion whereas prevalent binding close to an alternative site often causes exon skipping. This positional effect was further demonstrated by disrupting or creating a PTB binding site on minigene constructs and testing their responses to PTB knockdown or overexpression. These findings suggest a mechanism for PTB to modulate splice site competition to produce opposite functional consequences, which may be generally applicable to RNA binding splicing factors to positively or negatively regulate alternative splicing in mammalian cells. Examination of PTB-RNA binding in Hela cells using CLIP-seq (Cross-Linking ImmunoPrecipitation coupled with high-throughput sequencing) method. Peaks: The four alignment files (linked as supplementary files on Sample records) were combined together for peak finding, as we found that most of the monomeric and dimeric tags are similarly distributed in the genome with high pearson correlation coefficient. The method to detect the peaks above gene-specific randomized background was similar to (Yeo et al., 2009) and described in the paper (Xue et al., 2009).
Project description:A core task to understand the consequences of non-coding single nucleotide polymorphisms (SNP) is to identify their genotype specific binding of transcription factor (TF). Here, we generate a large-scale TF-SNP interaction map for a selection of 116 colorectal cancer (CRC) risk loci and validated TF binding to 10 putatively functional SNPs. Our data further revealed TF binding complexity adjacent to the 116 risk loci, adding an additional layer of understanding to regulatory networks associated with CRC relevant loci.
Project description:A focussed microarray chip was constructed specifically for studying basal colonic gene expression in patients with Spondyloarthropathy (SpA) and Crohn's disease (CD) (ref: PMID 16476712). These focus microarrays were used 1) to study common changes in gene expression between SpA and CD, providing early markers in the follow-up study of patients with SpA and particularly these patients that evolve into clinically overt CD; and 2) to identify new candidate genes for CD by integrating gene expression data with genetic information. Keywords: Crohn's Disease, Spondylarthropathy, biopsies This experiment was done in a dye-swap design in which all samples of interest were compared to a pooled sample.
Project description:The nuclear matrix associated hnRNP U/SAF-A protein has been implicated in diverse pathways from transcriptional regulation to telomere length control to X inactivation, but the precise mechanism underlying each of these processes has remained elusive. Here, we report hnRNP U as a regulator of SMN2 splicing from a custom RNAi screen. Genome-wide analysis by CLIP-seq reveals that hnRNP U binds virtually to all classes of regulatory non-coding RNAs, including all snRNAs required for splicing of both major and minor classes of introns, leading to the discovery that hnRNP U regulates U2 snRNP maturation and Cajal body morphology in the nucleus. Global analysis of hnRNP U-dependent splicing by RNA-seq coupled with bioinformatic analysis of associated splicing signals suggests a general rule for splice site selection through modulating the core splicing machinery. These findings exemplify hnRNP U/SAF-A as a potent regulator of nuclear ribonucleoprotein particles in diverse gene expression pathways. Examination of hnRNP U regulated splicing in Hela cells with CLIP-seq (two biological replicates) and paired-end RNA-seq (control and hnRNP U knockdown)
Project description:Trans-splicing occurs post-transcriptionally and generates transcripts that are orderly inconsistent with their corresponding DNA templates. Until recently only exceedingly rare trans-splicing events have been experimentally characterized in the mammalian transcriptomes. Although hundreds to thousands of trans-spliced RNA candidates have been nominated by bioinformatics- or NGS (next-generation sequencing)-based approaches, these candidates unavoidably suffered from potential false positives arising from genetic rearrangement events or in vitro artifacts. Here we develop a pipeline (TSscan) based on NGS transcriptome data to identify trans-splicing in human embryonic stem cells (ESCs). TSscan integrates RNA sequencing data derived from different NGS platforms (i.e., Roche 454, SOLiD, and Illumina) and different human ESC lines (i.e., H1 and H9) as well as several in silico filters to minimize these two types of potential false positives. Our result shows that a tremendous amount of apparent experimental artifacts are indeed present in NGS data, which may be the most major false positives of trans-splicing detection. TSscan totally identified 10 trans-spliced RNA candidates in human ESCs, four of which are experimentally validated to be true. Further experiments reveal that these four events represent differential expression during the transition of pluripotent status to differentiate statuses. Especially, we observe that one event (the trans-spliced isoform of NCRMS), which is also a large intergenic non-coding RNA, tends to be specifically transcribed in ESCs and induced pluripotent stem cells and can conspicuously affect the pluripotency maintenance of ESCs. As far as we know, TSscan is the first pipeline for systematic identification of trans-splicing that utilizes NGS data in the human transcriptome, opening up an important class of post-transcriptional events for comprehensive characterization. human embryonic stem cell H9
Project description:RNA-seq was performed on encysting and excysting E. invadens, a parasite of reptiles that is used as a model system to study stage conversion in Entamoeba species, including the human pathogen E. histolytica. The goal of the project was to identify changes to the transcriptome during development, in order to better understand the mechanisms of development. RNA was isolated from trophozoites, from encysting parasites (at 8, 24, 48 and 72 hours after transfer to encystation media) and from excysting parasites (2 and 8 hours after induction of excystation), converted to cDNA, and sequenced on a SOLiD platform. RNA-seq data was used to refine gene models, find potential unannotated genes, and identify genes that are developmentally regulated.
Project description:Individual-nucleotide resolution UV-crosslinking and immunoprecipitation (iCLIP) combined with high-throughput sequencing was used to generate a transcriptome-wide binding map of hnRNP L. Supplementary file GSE37560_hnRNPL_crosslink_site.bed includes filtered crosslink sites of hnRNPL: combining data from all 3 experiments. 3 biological replicates of hnRNP L-specific and control (Flag) co-immunoprecipitated RNA after UV-crosslinking in HeLa cells