PCR cycles affect gibbs free energy bias
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE149914 | GEO | 2020/05/06
REPOSITORIES: GEO
Similar Datasets
Project description:PCR cycles affect gibbs free energy bias
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Project description:As transposon sequencing (TnSeq) assays have become prolific in the microbiology field, it is of interest to scrutinize their potential drawbacks. TnSeq results are determined by counting transposon insertions following the PCR-based enrichment and subsequent deep sequencing of transposon insertions. Here we explore the possibility that PCR amplification of transposon insertions in a TnSeq library skews the results by introducing bias into the detection and/or enumeration of insertions. We compared the detection and frequency of mapped insertions when altering the number of PCR cycles in the enrichment step. In addition, we devised and validated a novel, PCR-free TnSeq method where the insertions are enriched via CRISPR/Cas9-targeted transposon cleavage and subsequent Oxford Nanopore sequencing. These PCR-based and PCR-free experiments demonstrate that, overall, PCR amplification does not significantly bias the results of the TnSeq assay insofar as insertions in the majority of genes represented in our library were similarly detected regardless of PCR cycle number and whether or not PCR amplification was employed. However, the detection of a small subset of genes which had been previously described as essential is indeed sensitive to the number of PCR cycles. We conclude that PCR-based enrichment of transposon insertions in a TnSeq assay is reliable but researchers interested in profiling essential genes should carefully weigh the number of amplification cycles employed in their library preparation protocols. In addition, we present a PCR-free TnSeq alternative that is comparable to traditional PCR-based methods although the latter remain superior owing to their accessibility and high sequencing depth.
2020-12-21 | GSE162988 | GEO
Project description:We infected ZNF417 ZNF587 dKO 293T or control 293T with NL4.3 virus for 48 hours. Then genomic DNA was purified from cell lysis with by phenol chloroform. DNA was sheared into 300 bp-500 bp random fragments using Covaris Adaptive Focused Acoustics (Covaris, Woburn, MA). Sheared DNA was ligated with a linker and PCR with primers targeting HIV LTR and linker. PCR products were performed second PCR with Illumina adaptor primers. Libraries were so constructed and sequenced on Novaseq 6000.
2021-12-31 | GSE174239 | GEO
Project description:Budding yeast grown under continuous, nutrient-limited conditions exhibit robust, highly periodic cycles in the form of respiratory bursts. Microarray studies reveal that over half of the yeast genome is expressed periodically during these metabolic cycles. Genes encoding proteins having a common function exhibit similar temporal expression patterns, and genes specifying functions associated with energy and metabolism tend to be expressed with exceptionally robust periodicity. Essential cellular and metabolic events occur in synchrony with the metabolic cycle, demonstrating that key processes in a simple eukaryotic cell are compartmentalized in time. This data set contains the raw affymetrix gene expression data over three successive metabolic cycles. 12 time intervals per cycle, ~25 min per time interval.
2007-06-24 | E-GEOD-3431 | biostudies-arrayexpress
Project description:ChIP-seq analysis was used to identify genes potentially regulated by BMAL1 in undifferentiated (UNDIFF) and lactogen hormone differentiated (DIFF) HC11 cultures. HC11 cells are a normal mouse mammary epithelial cell line. Prior to beginning studies, specificity of the ChIP-grade antibody for the BMAL1 protein was confirmed with immunoprecipitation and western blot analysis. Nanochip analysis of sonicated input DNA indicated that optimal size of sheared fragments was achieved for ChIP-seq analysis. Moreover, ChIP-qPCR analysis of the promoter region of PER1, a transcriptional target of BMAL1:CLOCK, found that 70% of the input DNA sequence was captured by chromatin immunoprecipitation, with the q-PCR product being 9-fold higher in BMAL1-ChIP samples than mock samples (no primary antibody added). Q-PCR analysis of the exon region of MAGEA 1_2, a sperm specific gene that is not a BMAL1:CLOCK target was used as a negative control. There was no difference in amount of MAGEA 1_2 q-PCR between mock-ChIP and BMAL1-ChIP samples, thus further validating the BMAL1-ChIP assay. The eight samples sequenced for these studies consisted of four pairs of input and ChIP samples with two pairs from UNDIFF and two from DIFF HC11 cultures. Mapping rate of reads to the mouse genome across the four paired input-ChIP samples averaged 96%.
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Project description:Genome wide rhythmic transcription under light/dark cycles is associated with sequential transcription of specific biological processes genes in Ostreococcus tauri. Transcriptional profiling of Ostreococcus tauri under light/dark cycles. In order to identify genes with a diurnal rhythm, cells entrained under 12:12 light/dark cycles were sampled every 3 hours for 27 hours with two overlapping time points at Time 9 (Light ON at Time 9; Light OFF at Time 21) in 3 independent experiments.
2010-04-20 | GSE16422 | GEO
Project description:Mitochondria are major suppliers of cellular energy through nutrients oxidation. Little is known about the mechanisms that enable mitochondria to cope with changes in nutrient supply and energy demand that naturally occur throughout the day. To address this question, we applied mass-spectrometry based quantitative proteomics on isolated mitochondria from mice sacrificed throughout the day and identified extensive oscillations in the mitochondrial proteome. We found that rate-limiting mitochondrial enzymes that process lipids and carbohydrates accumulate in a diurnal manner and dependent on the clock proteins PER1/2. In this conjuncture, we uncovered daily oscillations in mitochondrial respiration that peak during different times of the day in response to different nutrients. Notably, the diurnal regulation of mitochondrial respiration was blunted in mice lacking PER1/2 or upon nutritional challenge such as high fat diet. We propose that the clock proteins PER1/2 optimize mitochondrial metabolism to daily changes in energy supply/demand and thereby serve as a rheostat for mitochondrial nutrient utilization.
2018-10-17 | PXD001732 | Pride
Project description:Investigating PCR bias in TnSeq
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Project description:It is well known that host-microbes and immunity interactions are influenced by dietary patterns, as well as daily environmental light-dark (LD) cycles that entrain circadian rhythms in the host. Emerging data has highlighted the importance of diet patterns and timing on the interaction among circadian rhythms, gut microbiome, and immunity, however, their impacts on LD cycles are less reported. Therefore, we aim to study how LD cycles regulate the homeostatic crosstalk between gut microbiome, hypothalamic and hepatic circadian clock oscillations and immunity. We hypothesized that different environmental LD cycles: (1) constant darkness, LD0/24; (2) short light, LD8/16; (3) normal LD cycle, LD12/12; (4) long light, LD16/8; and (5) constant light, LD24/0, may affect immunity and metabolism to varying degrees. Therefore, 240 mice were managed with chow diets (CD) and antibiotics treatments (ABX) under five different LD cycles for 42 days. The colonic (co) and cecum (ce) contents were obtained for studying their impacts on gut microbiome using 16S rRNA sequencing.
2023-03-10 | GSE226852 | GEO
Project description:Insects use diapause, a programmed period of dormancy, to avoid stressful times of the year and to exploit seasonal times of resource availability. Because most diapausing insects do not feed, they must live off their body reserves for several months and the proper use of metabolic reserves is critical for surviving diapause and performing after diapause termination. Across multiple insects, metabolic depression during diapause has been associated with a switch from aerobic metabolism to facultative anaerobic metabolism, despite insects not suffering environmental oxygen limitation. While metabolic rates are depressed during diapause overall to save energy, some insects show regular cyclical bouts of higher metabolic activity during diapause. The functional importance of these metabolic cycles and the mechanisms underlying these cycles are still unknown, but they may be critical for properly maintaining the balance between energy states and purge the accumulation of anaerobic metabolic byproducts. In the present study, we will test the hypothesis that periodic cycles of increased metabolism during insect diapause are associated with both regenerating organismal energetic states, particularly ATP that may decline during metabolic depression, and for purging metabolites associated with anaerobic metabolism. We will use a combination of non-targeted uHPLC-MS/MS metabolomics and targeted NMR-spectroscopy to identify and quantify metabolites that are altered during the cycles in diapausing pupae of the flesh fly, Sarcophaga crassipalpis. This work will allow us to propose specific biochemical and cellular hypotheses for the regulation of cyclic releases from metabolic depression in diapausing insects. Our work may not only reveal the physiological mechanisms regulating metabolic cycles during diapause in flesh fly, but also provide insight to understand the regulation of similar metabolic cycles in mammalian hibernators (i.e., periodic arousal), and also provide insights into how these cycles could be exploited to disrupt the diapause of insect pests.
2015-03-03 | ST000294 | MetabolomicsWorkbench