Project description:The submitted files contain ChIP-seq data for the p300 transcriptional coactivator in GM12878 cells and for the NRSF transcription factor in GM12878 and Jurkat cells generated using a fully automated robotic chromatin immunoprecipitation protocol. Cells were fixed using 1% formaldehyde (NRSF samples) or 1% formaldehyde at 37C (p300 samples).
Project description:Crucial conventional patch-clamp approaches to investigate cellular electrophysiology suffer from low-throughput and require considerable experimenter expertise. Automated patch-clamp (APC) approaches are more experimenter independent and offer high-throughput, but by design are predominantly limited to assays containing small, homogenous cells. In order to enable high-throughput APC assays on larger cells such as native cardiomyocytes isolated from mammalian hearts, we employed a fixed-well APC plate format. A broad range of detailed electrophysiological parameters including action potential, L-type calcium current and basal inward rectifier current were reliably acquired from isolated swine atrial and ventricular cardiomyocytes using APC. Effective pharmacological modulation also indicated that this technique is applicable for drug screening using native cardiomyocyte material. Furthermore, sequential acquisition of multiple parameters from a single cell was successful in a high throughput format, substantially increasing data richness and quantity per experimental run. When appropriately expanded, these protocols will provide a foundation for effective mechanistic and phenotyping studies of human cardiac electrophysiology. Utilizing scarce biopsy samples, regular high throughput characterization of primary cardiomyocytes using APC will facilitate drug development initiatives and personalized treatment strategies for a multitude of cardiac diseases.
Project description:The human enteroendocrine L cell line NCI-H716, expressing taste receptors and taste signaling elements, constitutes a unique model for the studies of cellular responses to glucose, appetite regulation, gastrointestinal motility, and insulin secretion. Targeting these gut taste receptors may provide novel treatments for diabetes and obesity. However, NCI-H716 cells are cultured in suspension and tend to form multicellular aggregates, preventing high-throughput calcium imaging due to interferences caused by laborious immobilization and stimulus delivery procedures. Here, we have developed an automated microfluidic platform that is capable of trapping more than 500 single cells into microwells with a loading efficiency of 77% within two minutes, delivering multiple chemical stimuli and performing calcium imaging with enhanced spatial and temporal resolutions when compared to bath perfusion systems. Results revealed the presence of heterogeneity in cellular responses to the type, concentration, and order of applied sweet and bitter stimuli. Sucralose and denatonium benzoate elicited robust increases in the intracellular Ca(2+) concentration. However, glucose evoked a rapid elevation of intracellular Ca(2+) followed by reduced responses to subsequent glucose stimulation. Using Gymnema sylvestre as a blocking agent for the sweet taste receptor confirmed that different taste receptors were utilized for sweet and bitter tastes. This automated microfluidic platform is cost-effective, easy to fabricate and operate, and may be generally applicable for high-throughput and high-content single-cell analysis and drug screening.
Project description:Autophagic processes play a central role in cellular homeostasis. In pathological conditions, the flow of autophagy can be affected at multiple and distinct steps of the pathway. Current analyses tools do not deliver the required detail for dissecting pathway intermediates. The development of new tools to analyze autophagic processes qualitatively and quantitatively in a more straightforward manner is required. Defining all autophagy pathway intermediates in a high-throughput manner is technologically challenging and has not been addressed yet. Here, we overcome those requirements and limitations by the developed of stable autophagy and mitophagy reporter-iPSC and the establishment of a novel high-throughput phenotyping platform utilizing automated high-content image analysis to assess autophagy and mitophagy pathway intermediates.
Project description:Setup and optimisation of a high throughput pipeline for ChIPseq. The protocol used is ChIP carried out using the Agilent Bravo robot with subsequent Ilumina sequencing library preparation.This data is part of a pre-publication release. For information on the proper use of pre-publication data shared by the Wellcome Trust Sanger Institute (including details of any publication moratoria), please see http://www.sanger.ac.uk/datasharing/
Project description:Permethylation is an essential technique for the detailed structural characterization of glycans by mass spectrometry. However, it requires specialized skills and equipment and is labor-intensive. While this limits glycan analysis to a handful of experts, the increasing awareness of the tremendous importance of glycans in physiological processes of health and disease has drastically raised the demand for detailed structural characterization of glycans. We have developed a simple microplate permethylation method that relies upon solid phase extraction using C18 tips to purify the permethylated glycans. The procedure is easy to perform, making it accessible to nonexperts, and fast, promising to accelerate glycan research. A 96-well plate of released glycan samples can be permethylated in less than an hour. The procedure can be carried out without discontinuous steps in an automatic pipet and integrated seamlessly with glycan release and mass spectrometry.
Project description:The high-throughput screening of recombinant protein expression is advantageous during early process development because it allows the identification of optimal expression constructs and process conditions. Simple screening platforms based on microtiter plates are available for microbes and animal cells, but this was not possible for plants until the development of plant cell packs (PCPs), also known as "cookies," which provide a versatile and scalable screening tool for recombinant protein production. PCPs are prepared from plant cell suspension cultures by removing the medium and molding the biomass. PCPs can be cast into 96-well plates for high-throughput screening, but the manual handling effort currently limits the throughput to ∼500 samples per day. We have therefore integrated the PCP method with a fully automated laboratory liquid-handling station. The "robot cookies" can be prepared and infiltrated with Agrobacterium tumefaciens by centrifugation, minimizing operator handling and reducing the likelihood of errors during repeated runs, such as those required in a design of experiments approach. The accumulation of fluorescent protein in the cytosol, apoplast, endoplasmic reticulum or plastids is easily detected using an integrated plate reader, reducing the inter-experimental variation to <5%. We also developed a detergent-based chemical lysis method for protein extraction in a 96-well format, which was adapted for automated downstream processing using miniaturized columns allowing subsequent protein analysis. The new automated method reduces the costs of the platform to <0.5 € per PCP infiltration (a saving of >50%) and facilitates a five-fold increase in throughput to >2500 samples per day.
Project description:The zebrafish larva stands out as an emergent model organism for translational studies involving gene or drug screening thanks to its size, genetics, and permeability. At the larval stage, locomotion occurs in short episodes punctuated by periods of rest. Although phenotyping behavior is a key component of large-scale screens, it has not yet been automated in this model system. We developed ZebraZoom, a program to automatically track larvae and identify maneuvers for many animals performing discrete movements. Our program detects each episodic movement and extracts large-scale statistics on motor patterns to produce a quantification of the locomotor repertoire. We used ZebraZoom to identify motor defects induced by a glycinergic receptor antagonist. The analysis of the blind mutant atoh7 revealed small locomotor defects associated with the mutation. Using multiclass supervised machine learning, ZebraZoom categorized all episodes of movement for each larva into one of three possible maneuvers: slow forward swim, routine turn, and escape. ZebraZoom reached 91% accuracy for categorization of stereotypical maneuvers that four independent experimenters unanimously identified. For all maneuvers in the data set, ZebraZoom agreed with four experimenters in 73.2-82.5% of cases. We modeled the series of maneuvers performed by larvae as Markov chains and observed that larvae often repeated the same maneuvers within a group. When analyzing subsequent maneuvers performed by different larvae, we found that larva-larva interactions occurred as series of escapes. Overall, ZebraZoom reached the level of precision found in manual analysis but accomplished tasks in a high-throughput format necessary for large screens.
Project description:The use of capillary electrophoresis with fluorescently labeled nucleic acids revolutionized DNA sequencing, effectively fueling the genomic revolution. We present an application of this technology for the high-throughput structural analysis of nucleic acids by chemical and enzymatic mapping ('footprinting'). We achieve the throughput and data quality necessary for genomic-scale structural analysis by combining fluorophore labeling of nucleic acids with novel quantitation algorithms. We implemented these algorithms in the CAFA (capillary automated footprinting analysis) open-source software that is downloadable gratis from https://simtk.org/home/cafa. The accuracy, throughput and reproducibility of CAFA analysis are demonstrated using hydroxyl radical footprinting of RNA. The versatility of CAFA is illustrated by dimethyl sulfate mapping of RNA secondary structure and DNase I mapping of a protein binding to a specific sequence of DNA. Our experimental and computational approach facilitates the acquisition of high-throughput chemical probing data for solution structural analysis of nucleic acids.