Project description:Top-Down proteomics pilot experiment of unfractionated Bovine Heart Mitochondria (BHM) using ultra high resolution Q-ToF tandem mass spectrometry (maXis 4G ETD, Bruker Daltonics).
Project description:Purpose: Identification of glucocorticoid recepor binding sites on human kidney proximal tubular cells to better understand epigenetic alterations in diabetic kidney diseases Methods: CUT&RUN assay libraries for cultured cells or human kidneys were generated with the CUTANA kit (EpiCypher, 14-1048). 500,000 cells were mixed and incubated with Concanavalin A (ConA) conjugated paramagnetic beads. Antibodies to the glucocorticoid receptor were added to target samples and IgG was added to negative controls. The remaining steps were performed according to manufacturer’s instructions. Library preparation was performed using the NEBNext Ultra II DNA Library Prep Kit for Illumina (New England BioLabs, E7645S) with manufacturer’s instructions, including minor modifications indicated by CUTANA described above. CUT&RUN libraries were sequenced on a NovaSeq instrument (Illumina, 150 bp paired-end reads). For bulk ATAC-seq, 50,000 nuclei were transposed in 25 μL of ATAC-seq transposition mix [12.5 μL 2× Illumina Tagment DNA (TD) buffer; 10.5 μL nuclease-free water; 2.0 μL Tn5 transposase (Illumina/Nextera, FC-121-1030)] and incubated at 37°C for 1 h on a thermomixer. The transposed DNA was purified with QIAGEN MinElute kit (28004) and amplified with dual indexes.
Project description:Plasma samples of patients diagnosed with CCM. Samples were run with a standard extraction (Plate 1 5x) and then again through a Phree Kit (Phree Kit Plate) to remove phospholipids. Data was acquired using a Bruker Maxis Impact and C18 RP-UHPLC using positive and negative polarity of LC-MS/MS.
Project description:Data provided report the use of trapped ion mobility spectrometry (TIMS) to fractionate ions in the gas phase based on their ion mobility (V⋅s/cm2) followed by parallel accumulation serial fragmentation (PASEF) in a quadrupole time-of-flight instrument. TIMS fractionation coupled to DDA-PASEF allowed for the detection of approximately 7,000 proteins and over 70,000 peptides per overall run from 200ng of human (HeLa) cell lysate per injection using a commercial UPLC column with a 90-minute gradient. This project also explored the utility of TIMS fractionation to generate a DDA library for downstream DIA analysis using shorter LC gradients (20 minutes) as well as lower sample input. Using a 20min gradient, we identified 4,092 and 6,654 proteins on average per run, respectively, from 10ng and 200ng of human (HeLa) cell lysate input based on a TIMS-fractionated library consisting of 82,214 peptides derived from 7,615 proteins.
Project description:To study the function of Physcomitrella DCL4, a knockout plant line was created. Small RNAs from ~10-day old protonemata were obtained and sequenced using a SOLiD 2 instrument. A single library each was made from dcl4 mutant protonemata and wild-type protonemata. The dcl4 library was sequenced in four separate technical replicates. The wild-type sample was subject to a single sequencing run.
Project description:Creation of a new library entries for Candida auris using MALDI Biotyper. Candida auris has a high genetic variability in the world, the identification of Colombian isolates is difficult using the main Bruker library. A new in-house library was created using Colombian isolated and was validated using 300 isolated strains
Project description:Different sample preparation methods were tested for HeLa proteome analysis. A sample obtained using sodium deoxycholate-based lysis allowed identification of the highest number of proteins. For this sample, a dilution series was acquired in triplicates ranging from 0.2ng to 200ng. All measurements were performed on Bruker timsTOF Pro 2 operated in dia-PASEF mode and analysed library-free using DIA-NN 1.8.
Project description:The project aimed to characterize the collagen type I (COL1) sequences from Pleistocene Macrauchenia sp. and Toxodon sp. bone samples, and by comparison with existing COL1 sequences available from genomic sources establish the phylogenetic position of both extinct species. In order to resolve their phylogenetic position, COL1 was extracted from two Toxodon (samples MLP201204, MACN201212, York12, York13) and two Macrauchenia (samples MLP201212, MACN201202, York14, York15). In addition, modern and Pleistocene COL1 was extracted from additional species currently not present in available databases (Mylodon darwinii, Cyclopes didactylus, Hippopotamus amphibius, Tapirus terrestris) or from species for which COL1 sequences are available (Equus sp., Oryceropus afer). All extractions were performed at BioArCh, University of York (UK). Analyses took place on Bruker maXis HD (Macrauchenia sp., Toxodon sp., Equus sp.) and Thermo Scientific Hybrid Quadruopole-Orbitrap (Macrauchenia sp., Toxodon sp., Mylodon darwinii, Cyclopes didactylus, Hippopotamus amphibius, Tapirus terrestris, Oryceropus afer) platforms.
Project description:Population scale sweeps of viral pathogens, such as SARS-CoV-2, require high intensity testing for effective management. However, reliable systems affording parallel testing of thousands of patients for pathogen infection have not yet been routinely employed. Here we describe “Systematic Parallel Analysis of RNA coupled to Sequencing for Covid-19 screening” (C19-SPAR-Seq), a multiplexed, readily automated platform for SARS-CoV-2 detection capable of analyzing tens of thousands of patient samples in a single instrument run. To address strict requirements for control of assay parameters and output demanded by clinical diagnostics, we employed a control-based Precision-Recall and Receiver Operator Characteristics (coPR) analysis to assign run-specific quality control metrics. C19-SPAR-Seq coupled to coPR on a trial cohort of several hundred patients performed with a specificity of 100% and sensitivity of 91% on samples with low viral loads. Our study thus establishes the feasibility of employing C19-SPAR-Seq for the large-scale monitoring of SARS-CoV-2 and other pathogens.
Project description:Population scale sweeps of viral pathogens, such as SARS-CoV-2, require high intensity testing for effective management. However, reliable systems affording parallel testing of thousands of patients for pathogen infection have not yet been routinely employed. Here we describe “Systematic Parallel Analysis of RNA coupled to Sequencing for Covid-19 screening” (C19-SPAR-Seq), a multiplexed, readily automated platform for SARS-CoV-2 detection capable of analyzing tens of thousands of patient samples in a single instrument run. To address strict requirements for control of assay parameters and output demanded by clinical diagnostics, we employed a control-based Precision-Recall and Receiver Operator Characteristics (coPR) analysis to assign run-specific quality control metrics. C19-SPAR-Seq coupled to coPR on a trial cohort of several hundred patients performed with a specificity of 100% and sensitivity of 91% on samples with low viral loads. Our study thus establishes the feasibility of employing C19-SPAR-Seq for the large-scale monitoring of SARS-CoV-2 and other pathogens.