ABSTRACT
Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is a method used to profile protein-DNA interactions genome-wide. Restriction Enzyme-based Labeling of Chromatin in Situ (RELACS) is a recently developed ChIP-seq protocol that deploys a chromatin barcoding strategy to enable standardized and high-throughput generation of ChIP-seq data. The manual implementation of RELACS is constrained by human processivity in both data generation and data analysis. To overcome these limitations, we have developed AutoRELACS, an automated implementation of the RELACS protocol using the liquid handler Biomek i7 workstation. We match the unprecedented processivity in data generation allowed by AutoRELACS with the automated computation pipelines offered by snakePipes. In doing so, we build a continuous workflow that streamlines epigenetic profiling, from sample collection to biological interpretation. Here, we show that AutoRELACS successfully automates chromatin barcode integration, and is able to generate high-quality ChIP-seq data comparable with the standards of the manual protocol, also for limited amounts of biological samples.
Subject(s)
Chromatin Immunoprecipitation/methods , Sequence Analysis, DNA/methods , Automation , Cell CountABSTRACT
MOTIVATION: This paper presents Parkour, a software package for sample processing and quality management of next generation sequencing data and samples. RESULTS: Starting with user requests, Parkour allows tracking and assessing samples based on predefined quality criteria through different stages of the sample preparation workflow. Ideally suited for academic core laboratories, the software aims to maximize efficiency and reduce turnaround time by intelligent sample grouping and a clear assignment of staff to work units. Tools for automated invoicing, interactive statistics on facility usage and simple report generation minimize administrative tasks. Provided as a web application, Parkour is a convenient tool for both deep sequencing service users and laboratory personal. A set of web APIs allow coordinated information sharing with local and remote bioinformaticians. The flexible structure allows workflow customization and simple addition of new features as well as the expansion to other domains. AVAILABILITY AND IMPLEMENTATION: The code and documentation are available at https://github.com/maxplanck-ie/parkour. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Subject(s)
High-Throughput Nucleotide Sequencing , Software , Humans , Laboratories , WorkflowABSTRACT
MOF (MYST1, KAT8) is the major H4K16 lysine acetyltransferase (KAT) in Drosophila and mammals and is essential for embryonic development. However, little is known regarding the role of MOF in specific cell lineages. Here we analyze the differential role of MOF in proliferating and terminally differentiated tissues at steady state and under stress conditions. In proliferating cells, MOF directly binds and maintains the expression of genes required for cell cycle progression. In contrast, MOF is dispensable for terminally differentiated, postmitotic glomerular podocytes under physiological conditions. However, in response to injury, MOF is absolutely critical for podocyte maintenance in vivo. Consistently, we detect defective nuclear, endoplasmic reticulum and Golgi structures, as well as presence of multivesicular bodies in vivo in podocytes lacking Mof following injury. Undertaking genome-wide expression analysis of podocytes, we uncover several MOF-regulated pathways required for stress response. We find that MOF, along with the members of the non-specific lethal but not the male-specific lethal complex, directly binds to genes encoding the lysosome, endocytosis and vacuole pathways, which are known regulators of podocyte maintenance. Thus, our work identifies MOF as a key regulator of cellular stress response in glomerular podocytes.
Subject(s)
Histone Acetyltransferases/genetics , Stress, Physiological/genetics , Animals , Cell Cycle Checkpoints/genetics , Heat-Shock Proteins/genetics , Heat-Shock Proteins/metabolism , Histone Acetyltransferases/metabolism , Humans , Mice , Mice, Inbred C57BL , Mice, Transgenic , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Podocytes/cytology , Podocytes/metabolism , Podocytes/physiology , Scavenger Receptors, Class A/genetics , Scavenger Receptors, Class A/metabolism , Transcription, GeneticSubject(s)
Dermatitis/genetics , Desmoglein 1/genetics , Hypersensitivity/genetics , Mutation/genetics , Wasting Syndrome/genetics , Adult , Age of Onset , Child , Desmoglein 1/deficiency , Exome , Genes, Recessive , Genome-Wide Association Study , Humans , Keratoderma, Palmoplantar/genetics , Norway , Pedigree , SyndromeABSTRACT
Light quark masses are calculated in lattice QCD with two degenerate flavors of dynamical quarks. The calculations are made with improved actions with lattice spacing a = 0.22-0.11 fm. In the continuum limit we find m(M&Smacr;)(ud)(2 GeV) = 3.44(+0.14)(-0.22) MeV using the pi and rho meson masses as physical input, and m(M&Smacr;)(s)(2 GeV) = 88(+4)(-6) MeV or 90(+5)(-11) MeV with the K or straight phi meson mass as additional input. The quoted errors represent statistical and systematic combined, the latter including those from continuum and chiral extrapolations, and from renormalization factors. Compared to quenched results, two flavors of dynamical quarks reduce quark masses by about 25%.
ABSTRACT
A survey for Arcobacter spp. and Arcobacter butzleri in mechanically separated turkey was conducted during the winter of 1995 and summer and fall of 1996. Arcobacter spp. and A. butzleri were identified by polymerase chain reaction and species-specific oligonucleotide probes. Arcobacter spp. were isolated from 77% (303 out of 395) of the mechanically separated turkey samples with 74% (223 out of 303) of these samples positive for A. butzleri. Of the 121 A. butzleri isolates tested, 86 different patterns were evident following amplification of enterobacterial repetitive intergenic consensus sequences. The extent of genetic polymorphism indicated multiple sources of contamination.
Subject(s)
Food Handling , Genetic Variation , Gram-Negative Bacteria/classification , Gram-Negative Bacteria/isolation & purification , Meat/microbiology , Turkeys/microbiology , Animals , Blotting, Southern , DNA Fingerprinting , DNA, Bacterial/analysis , Food Microbiology , Gram-Negative Bacteria/genetics , Nucleic Acid Hybridization , Polymerase Chain Reaction/methods , SeasonsABSTRACT
Arcobacter spp. have recently been genetically differentiated as a genus distinct from Campylobacter. Physiologically, Arcobacter spp. are aerotolerant bacteria, while Campylobacter spp. are microaerophilic. However, since Arcobacter spp. have been difficult to grow to high population densities in broth media, alternative culture techniques were investigated. A biphasic culture system was developed in 25 cm2 tissue culture flasks. Biphasic culture, consisting of a solid phase of 10% bovine blood agar and a liquid phase of Brain Heart Infusion broth, was found to increase bacterial population densities by more than 2 log10 cycles for strains of A. butzleri and A. skirrowii. A strain of A. cryaerophilus, which was non-culturable in broth culture, attained population densities of 10(9) cells ml-1 in biphasic culture. Neither the addition of fetal bovine serum to the liquid nor an increase in the surface area from 25 to 75 cm2 resulted in increased cell densities.
