The brain endothelial cell translatome analysis in the visual cortex at plastic and nonplastic ages
Ontology highlight
ABSTRACT: In this study, we provide the translatome data in the brain endothelial cells (BECs) of the visual cortex at 3 different time points (pre-critical period (P10), critical period (P30), post-critical period (P100)) by using translating ribosome affinity purification (TRAP) combined with high-throughput RNA sequencing analysis in Tie2-Cre:Ribo-Tag mice, which are designed for expressing hemagglutinin A (HA)-tagged RPL22, a ribosomal protein in endothelial cells depending on CRE recombinase. To obtain a high-quality BEC pool, we modified the TRAP method by adding the capillary isolation process before the immunoprecipitation step and finally verified BEC-specific translating mRNA purification using RT-PCR.
Project description:In this study, we provide the translatome data in the brain endothelial cells (BECs) of the adult cortex in response to acute neuronal activation by using translating ribosome affinity purification (TRAP) combined with high-throughput RNA sequencing analysis in Tie2-Cre:Ribo-Tag mice, which are designed for expressing hemagglutinin A (HA)-tagged RPL22, a ribosomal protein in endothelial cells depending on CRE recombinase. To increase neural activity rapidly, we adopted an acute epilepsy induction model in which pentylenetetrazol (PTZ), a GABAA receptor antagonist was injected and observed significant upregulation of c-fos-positive cells in the cortex. To obtain a high-quality BEC pool, we modified the TRAP method by adding the capillary isolation process before the immunoprecipitation step and finally verified BEC-specific translating mRNA purification using RT-PCR. We then analyzed RNA-seq data obtained from the BECs of cortex of adult mice injected with phosphate buffered saline (PBS) or PTZ and identified brain endothelial cell genes that are changed dynamically due to neuronal activation. This information will be useful for researchers to study neurovascular interaction or to identify biomarkers in epilepsy.
Project description:To identify transcripts enriched in ventomedial hypothalamus (VMH) neurons, we used translating affinity ribosome purification combined with RNA-seq (TRAP-seq) from Nr5a1-Cre mice.
Project description:To identify transcripts enriched in ventomedial hypothalamus (VMH) Lepr neurons, we used translating affinity ribosome purification combined with RNA-seq (TRAP-seq) from Lepr-Cre mice.
Project description:We profiled the gene expression patterns of undisturbed endothelial cells in living animals using a novel ‘AngioTag’ zebrafish transgenic line that permits isolation of actively translating mRNAs from endothelial cells in their native environment. This transgenic line uses the endothelial cell-specific kdrl promoter to drive expression of an epitope tagged Rpl10a 60S ribosomal subunit protein, allowing for Translating Ribosome Affinity Purification (TRAP) of actively translating endothelial cell mRNAs. We also collected the whole embryo translatome using the TRAP protocol and a ubiquitously expressed tagged Rpl10a, and the endothelial transcritome by collecting the GFP+ endothelial cells that had the kdrl promoter driving GFP.
Project description:To identify transcripts enriched in ventomedial hypothalamus (VMH) neurons that contain both Lepr and Slc17a6, we used translating affinity ribosome purification combined with RNA-seq (TRAP-seq) from Lepr-Cre;Slc17a6-Flpo mice.
Project description:PRV-Circuit-TRAP of DAT-cre mice injected with PRV-Introvert-GFP in the nucleus accumbens These studies identify important inputs to the mesolimbic dopamine pathway and further show that PRV circuit-directed translating ribosome affinity purification (PRV-Circuit-TRAP) can be broadly applied to identify molecularly defined neurons comprising complex, multisynaptic circuits.
Project description:To understand the transcriptional effect of fasting and feeding a ketogenic diet on mouse CNS astrocytes, we performed translating ribosomal affinity purification (TRAP) of mRNAs immunoprecipitated from hippocampus. TRAP mice express a ribosomal epitope tag upon Cre-induced recombination that can be immunoprecipitated following activation. We measured the abundance of actively translating mRNAs from a ribosomal pull-down that came from adult astrocyte (Aldh1l1-Cre)-specific TRAP mice that were subjected to one of three dietary conditions: four weeks of normal chow diet, four weeks of ketogenic diet (high-fat, low-carbohydrate)43, or an 18-hour fast. Immediately following the respective diets, forebrain and hippocampus was harvested from all groups, ribosomes were immunoprecipitated, and actively translating mRNAs in the ribosomes were purified.
Project description:We isolated RNAs from Otp-lineage cells in the mouse brain using translating ribosome affinity purification (TRAP) approach. We compared two groups of mice: Otp-cre; ROSAfsTRAP (control) and Otp-cre; Cicf/f; ROSAfsTRAP (conditional knockout). Following RNA isolation, RNAseq was performed and gene expression profiles were compared between controls and conditional knockouts.
Project description:Purpose: To generate gene expression profiles of inguinal white, epididymal white and interscapular brown adipocytes Methods: Translating ribosomal affinity purification (TRAP) using an adipocyte-specific cre in adult wildtype mice followed by RNA-Seq Results: Using an optimized data analysis workflow, we mapped about 30 million sequence reads per sample to the mouse genome (build mm9) using Tophat and assembled reads into transcripts using Cufflinks.
Project description:L1CAM dependent gene expression programs in vitro and ex vivo were analyzed by translating ribosome affinity purification and sequencing (TRAP-Seq).