Project description:Single cell five-prime end sequencing of PBMC - resting and stimulated. Used to asses the five-prime sequencing method in the detection of cis-regulatory elements using SCAFE (see publication)
Project description:Single cell ATAC-seq of PBMC - resting and stimulated. Used for comparison to asses the capabilies of the five-prime sequencing method in the detection of cis-regulatory elements using SCAFE (see publication).
Project description:The aim of the present project was to study in detail the site-specific phosphorylation events occurring both in resting as well as in T lymphocytes stimulated with IL-2 for five minutes. For that purpose we combined SILAC-based quantitative mass spectrometry analysis with phosphopeptide enrichment using TiO2 beads. We performed three biological replicas of the same experiment which resulted in the identification of above 8500 unique phosphosites corresponding to more than 3000 proteins. From the 6145 phosphosites that were consistently quantified in at least 2 out of the 3 replicas performed, we detected that 390 were regulated by IL-2 being the up-regulated phosphosites five times more abundant than the down-regulated ones. Those IL-2-dependent phosphosites corresponded to distinct proteins involved in distinct aspects of gene expression and cell cycle regulation.
Project description:We undertook gene expression microarray experiments to identify genes that are differentially expressed in invasive (Chorionic Girdle) and non-invasive (Chorion) placental tissue, and resting and Pokeweed Mitogen (PWM) stimulated horse lymphocytes. Conceptus tissues were dissected to obtain chorionic girdle, and chorion. Freshly isolated horse peripheral blood lymphocytes were split and harvested immediately, or stimulated with PWM and harvested over a five day period. These experiments utilized a commercially available Agilent horse array that featured >43,000 probes on a 4x44k array format. Three day 33-35 chorionic girdle RNAs were compared to matching chorion RNAs. Gene expression in resting lymphocytes was compared to gene expression in PWM treated lymphocytes.
Project description:The aim of the present project was to study in detail the site-specific phosphorylation events occurring both in resting as well as in T lymphocytes stimulated with IL-2 for five minutes. For that purpose we combined SILAC-based quantitative mass spectrometry analysis with phosphopeptide enrichment using TiO2 beads. We performed three biological replicas of the same experiment which resulted in the identification of above 8500 unique phosphosites corresponding to more than 3000 proteins. From the 6145 phosphosites that were consistently quantified in at least 2 out of the 3 replicas performed, we detected that 390 were regulated by IL-2 being the up-regulated phosphosites five times more abundant than the down-regulated ones. Those IL-2-dependent phosphosites corresponded to distinct proteins involved in distinct aspects of gene expression and cell cycle regulation.
Project description:We undertook gene expression microarray experiments to identify genes that are differentially expressed in invasive (Chorionic Girdle) and non-invasive (Chorion) placental tissue, and resting and Pokeweed Mitogen (PWM) stimulated horse lymphocytes. Conceptus tissues were dissected to obtain chorionic girdle, and chorion. Freshly isolated horse peripheral blood lymphocytes were split and harvested immediately, or stimulated with PWM and harvested over a five day period. These experiments utilized a commercially available Agilent horse array that featured >43,000 probes on a 4x44k array format.
Project description:As part of the zebrafish genome annotation project the 3 prime ends of genes were pulled down on polyT beads and sequenced on the Illumina Genome Analyzer to identify alternative 3 prime ends in a range of tissues and developmental stages. Total RNA from a range of developmental stages and adult tissues were chemically fragmented, pulled down on polyT magnetic beads and double stranded cDNA was synthesized. The cDNA was BpmI digested to release from the beads and to leave a 6 T base tail. Illumina sequencing libraries were made followed by 76 base paired-end sequencing. ArrayExpress Release Date: 2010-10-14 Person Roles: submitter Person Last Name: Collins Person First Name: John Person Mid Initials: E Person Email: jec@sanger.ac.uk Person Phone: 01233 834244 Person Address: Wellcome Trust Genome Campus, Hinxton, Cambridgeshire, CB10 1HH, United Kingdom Person Affiliation: Wellcome Trust Sanger Institute
Project description:Gene regulatory network analysis of resting and stimulated cells identifies a primed chromatin state within human monocytes [PBMC isolate scRNA-seq]
Project description:We undertook gene expression microarray experiments to identify genes that are differentially expressed in different placental and fetal tissue, and resting and Pokeweed Mitogen (PWM) stimulated horse lymphocytes. Conceptus tissues were dissected to obtain chorionic girdle, chorion, and fetal tissue. Freshly isolated horse peripheral blood lymphocytes were split and harvested immediately, or stimulated with PWM and harvested over a five day period. These experiments utilized a custom Agilent horse array designed in house that featured >14,000 probes on an 8x15k array format. Several genes were selected from the results for validation by quantitative real-time PCR. QPCR results matched the microarray results very closely. Four Day 33-35 chorionic girdle RNAs were compared to matching chorion RNAs, and fetal tissue from two of the conceptuses. Gene expression in resting lymphocytes was compared to gene expression in PWM treated lymphocytes.
Project description:Cells require coordinated control over gene expression changes when responding to environmental stimuli. Recent advancements in single-cell technologies have enabled studies of regulatory dynamics associated with cellular perturbation response in immune cells. However, associating these changes to underlying differences in epigenetic regulation through integration of multi-omic data has not been achieved at single-cell resolution. Here, we apply single-cell ATAC-seq (scATAC-seq) and scRNA-seq to assay chromatin accessibility and gene expression in resting and stimulated human blood cells. Collectively, we generate ~91,000 high-coverage single-cell profiles, allowing us to probe the cis-regulatory landscape of immunological response across cell types, stimuli and time. Advancing tools to integrate multiomic data, we connect distal cis-regulatory elements to genes, identifying key regulatory hubs associated with immune signaling and stress response. Subsequently, we design FigR - a method to infer gene regulatory networks (GRNs) to identify candidate TF regulators across single cells. Importantly, construction of a GRN using stimulus response data identifies TF regulatory activity associated with genetic variants in inflammatory diseases. Lastly, we assess the time-dependent regulatory dynamics of immune stimulation, where we find that cells alter chromatin accessibility prior to productive gene expression, a process occurring at time scales of minutes. Extending this concept of chromatin-mediated priming, and incorporating regulatory relationships defined by FigR, we detect a rare subpopulation of monocytes marked by a primed chromatin state predictive of enhanced immunological response. Overall, we build upon computational tools for GRN construction and demonstrate the utility of GRNs for analysis of multi-omic single cell data.