Systematic Evaluation of Parameters in RNA Cytosine Methylation Analysis
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ABSTRACT:
PROVIDER: PRJNA787668 | ENA |
REPOSITORIES: ENA
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Project description:Here we systematically analysis bisulfite-treated RNA sequencing data (RNA BS-seq) for the purpose of identifying m5C methylation data from mouse neural stem cells. We isolated mitochondrial fractions to better understand the m5C characteristics of RNAs found in these cellular compartments. Throughout the analysis, we identify key parameters to be used in RNA m5C methylation analysis, and identify downstream effects of artifact detection on RNA m5C levels. In addition, we utilized Unique Molecular Identifiers (UMIs) in conjunction with ERCC (External RNA Controls Consortium) as RNA controls to identify rates of m5C artifcats generated by RNA duplication and PCR amplification error rates. Using various preparation parameters, we identify ideal preparation methods for RNA BS-seq.
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Project description:Malaria parasites have evolved a well-adjusted developmental program to progress through the complex life cycle in the human and mosquito host. Here we identify cytosine methylation of tRNAAsp (GTC) as being critical to maintain stable protein synthesis under cellular stress. Using conditional knock out of a member of the DNA methyltransferases family called Pf-DNMT2, RNA bisulfite sequencing demonstrates the selective cytosine methylation of this enzyme of tRNAAsp (GTC) at position C38. Although no growth defect on parasite asexual proliferation was observed, Pf-DNMT2 KO parasites show a striking increase in their sexual commitment and an increased sensitivity to Dihydroartemisinin. In mutant parasites we observe a dramatic decrease of tRNAAsp (GTC) when compared to non-stressed parasites and mass spectrometry analysis shows the selective downregulation of proteins with GAC codon bias. Here we identify an epitranscriptomic mechanism that safeguards protein translation and homeostasis of sexual commitment during cellular stress in malaria parasites
2022-02-13 | PXD024769 | Pride
Project description:DNA methylation (5mC) plays important roles in epigenetic regulation of genome function, and recently the TET1-3 hydroxylases have been found to oxidize 5mC to hydroxymethylcytosine (5hmC), formylcytosine (5fC), and carboxylcytosine (5caC) in DNA. These derivatives have a role in demethylation of DNA but in addition may have epigenetic signaling functions in their own right. A recent study identified proteins with preferential binding to 5-methylcytosine (5mC) and its oxidized forms where readers for 5mC and 5hmC (5-hydroxymethylcytosine) showed little overlap while further oxidation forms enriched for repair proteins and transcription regulators. We extend this study by using promoter sequences as baits and compare protein binding patterns to unmodified or modified cytosine containing DNA using mouse embryonic stem cell (mESCs) extracts. The dataset contains 3 biological replicates each of mouse ES cell nuclear proteins binding to Pax6 and FGF15 promoter sequences containing different modified forms of cytosine. Data analysis: Mass spectrometric data were processed using Proteome Discoverer v1.3 and searched against a mammalian entries in Uniprot 2011.09 using Mascot v2.3 with the following parameters: Enzyme - trypsin; max 1 missed cleavage; Precursor Mass Tolerance - 10 ppm; Fragment Mass Tolerance - 0.6 Da; Dynamic Modification - Oxidation (M); Static Modification - Carbamidomethyl at C.
2013-10-21 | PXD000524 | Pride
Project description:To study if regulation of cytosine methylation and CTCF occupancy are interdependent and governed by the levels of CGGBP1, we specifically pulled-down methylated cytosines and found that some transcription factor binding sites including that of CTCF held out against the cytosine methylation changes. The cytosine methylation at CTCF-binding repeat-free motifs show a non-stochastic depdendence on CGGBP1 and occur at sites that mark cytosine methylation transition boundaries. We also show that allelic imbalance dictates stochastic methylation changes due to CGGBP1 depletion.
2020-07-20 | GSE145300 | GEO
Project description:The most widely used method for detecting genome-wide protein-DNA interactions is chromatin immunoprecipitation on tiling microarrays, commonly known as ChIP-chip. Here, we conducted the first objective analysis of tiling array platforms, amplification procedures, and signal detection algorithms in a simulated ChIP-chip experiment. Mixtures of human genomic DNA and spike-ins comprised of nearly 100 human sequences at various concentrations were hybridized to four tiling array platforms by eight independent groups. Blind to the number of spike-ins, their locations, and the range of concentrations, each group made predictions of the spike-in locations. We found that microarray platform choice is not the primary determinant of overall performance. In fact, variation in performance between labs, protocols and algorithms within the same array platform was greater than the variation in performance between array platforms. However, each array platform had unique performance characteristics that varied with tiling resolution and the number of replicates, which have implications for cost versus detection power. Long oligonucleotide arrays were slightly more sensitive at detecting very low enrichment. On all platforms, simple sequence repeats and genome redundancy tended to result in false positives. LM-PCR and WGA, the most popular sample amplification techniques, reproduced relative enrichment levels with high fidelity. Performance among signal detection algorithms was heavily dependent on array platform. The spike-in DNA samples and the data presented here provide a stable benchmark against which future ChIP platforms, protocol improvements, and analysis methods can be evaluated. This SuperSeries is composed of the following subset Series: GSE9732: Spike-in Experiment for ChIP-chip Simulation GSE9842: Systematic evaluation of variability in simulated ChIP-chip experiments GSE9848: Systematic evaluation of variability in simulated ChIP-chip experiments Kevin_Encode GSE9849: Systematic evaluation of variability in simulated ChIP-chip experiments Myles_Encode GSE10004: ENCODE Spike-In, Yale Group GSE10076: ENCODE spikein, amplified DNA samples, NimbleGen arrays GSE10090: ENCODE spikein, nonamplified DNA samples, NimbleGen arrays GSE10112: Systematic evaluation of variability in simulated ChIP-chip experiments Keywords: SuperSeries For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf Refer to individual Series
2010-09-30 | E-GEOD-10114 | biostudies-arrayexpress
Project description:Systematic evaluation of chromosome conformation capture assays [1D]
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Project description:Systematic evaluation of RNA-Seq preparation protocol performance
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Project description:Objectives: Formalin-fixed paraffin-embedded (FFPE) tissue is the standard material for di-agnostic pathology but poses relevant hurdles to accurate protein extraction due to cross-linking and chemical alterations. While numerous extraction pro-tocols and chemicals have been described, systematic comparative analyses are limited. Various parameters were thus investigated in their qualitative and quantitative effects on protein extraction (PE) efficacy. Special emphasis was put on preservation of membrane proteins (MP) as key subgroup of func-tionally relevant proteins. Methods: Using the example of urothelial carcinoma, FFPE tissue sections were subjected to various deparaffinization, protein extraction and antigen retrieval protocols and buffers as well as different extraction techniques. Performance was meas-ured by protein concentration and western blot analysis of cellular compart-ment markers as well as liquid chromatography-coupled mass spectrometry (LC-MS). Results: Commercially available extraction buffers showed reduced extraction of MPs and came at considerably increased costs. On-slide extraction did not improve PE whereas several other preanalytical steps could be simplified. Systematic variation of temperature and exposure duration demonstrated a quantitatively relevant corridor of optimal antigen retrieval. Conclusions: Preanalytical protein extraction can be optimized at various levels to improve unbiased protein extraction and to reduce time and costs.
2022-05-19 | PXD029133 | Pride