Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Ultrafast Bisulfite Sequencing for Efficient and Accurate 5-Methylcytosine Detection in DNA and RNA

ABSTRACT: We present an ultrafast bisulfite sequencing (UBS-seq) method, which accelerates the bisulfite reaction by about 13-fold, resulting in minimal DNA damage and much lower background in all DNA sequences. This ultrafast BS protocol is also ideal for mapping of RNA 5-methylcytosine (m5C) because of much shorter reaction time and dramatically reduced RNA background. And allowed detection of m5C stoichiometry in highly structured RNA species.

ORGANISM(S): Mus musculus Homo sapiens

PROVIDER: GSE225614 | GEO | 2023/10/13

REPOSITORIES: GEO

ACCESS DATA

Json Xml

Dataset's files

Source:

			Action	DRS
		Other

Items per page:

1 - 1 of 1

Similar Datasets

Accurate measurement of 5-methylcytosine and 5-hydroxymethylcytosine in human cerebellum DNA

Project description:The Infinium 450K Methylation array is an established tool for measuring methylation. However, the bisulfite (BS) reaction commonly used with the 450K array cannot distinguish between 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC). The oxidative-bisulfite assay disambiguates 5mC and 5hmC. We describe the use of oxBS in conjunction with the 450K array (oxBS-array) to analyse 5hmC/5mC in cerebellum DNA. The methylation level derived by the BS reaction is the combined level of 5mC and 5hmC at a given base, while the oxBS reaction gives the level of 5mC alone. The level of 5hmC is derived by subtracting the oxBS level from the BS level. Here we present an analysis method that distinguishes genuine positive levels of 5hmC at levels as low as 3 %. We preformed four replicates of the same sample of cerebellum and found a high level of reproducibility (average r for BS = 98.3, and average r for oxBS = 96.8). In total, 114,734 probes showed a significant positive measurement for 5hmC. The range at which we were able to distinguish 5hmC occupancy was between 3 % and 42 %. In order to investigate the effects of multiple replicates on 5hmC detection we also simulated fewer replicates and found that decreasing the number of replicates to two reduced the number of positive probes identified by > 50%. We validated our results using qPCR in conjunction with glucosylation of 5hmC sites followed by MspI digestion and we found good concordance with the array estimates (r = 0.94). This experiment provides a map of 5hmC in the cerebellum and a robust dataset for use as a standard in future 5hmC analyses. We also provide a novel method for validating the presence of 5hmC at low levels, and highlight some of the pitfalls associated with measuring 5hmC and 5mC.

2014-11-12 | GSE63179 | GEO

RNA 5-methylcytosine modification is enriched and conserved at 5' UTRs in human and mouse

Project description:Compared with the well-studied 5-methylcytosine (m5C) in DNA, the role of m5C in mRNA remains elusive as existing studies of RNA m5C are largely confined to rRNA and tRNA. Using bisulfite sequencing (BS-Seq) approach, we profiled transcriptome-wide m5C and found that RNA m5C modification is enriched and conserved at 5’UTRs in human and mouse. Through a comparative analysis of the mRNA and DNA methylome, we demonstrate that like DNA methylation, RNA m5C methylation exhibits a strong clustering. Surprisingly, mRNA Cytosine-5 methylation inversely correlates with DNA methylation under CpG context, which suggests a synergic regulatory effect. Importantly, comparison of the global RNA m5C methylome of breast cancer cells with normal mammary epithelial cells revealed that breast cancer methylome is predominantly hypomethylated and several breast cancer-related genes are differentially methylated. Despite global hypomethylation, 5’UTR of differentially methylated genes contain higher percentage of m5C. Taken together, this study provides a first-in-depth characterization of transcriptome-wide m5C modification. Furthermore, our findings showing differential m5C methylation pattern in normal and cancer cells suggest a vital role m5C methylation may play in the post transcriptional regulation of key pro-tumorigenic genes. Sample Preparation and RNA Bisulfite Sequencing: MCF10A and MDA-MB-468 (MDA468) cells were obtained from ATCC. Cells were cultured and maintained in DMEM media as per ATCC instructions. For BS-Seq total RNA was isolated from MCF10A and MDA_MB-468 cells and was enriched for polyA+ RNA using poly-A selection kits. The purified RNA is subjected to sodium bisulfite treatment at 60 degrees for 8 hours. The bisulfite-treated RNA was then reverse transcribed was subjected to deep-sequencing using Illumina RNA-Seq protocol.

2017-07-11 | GSE84230 | GEO

Base-resolution m5C profiling across the mammalian transcriptome by bisulfite-free enzyme-assisted chemical labeling approach

Project description:5-methylcytosine (m5C) is a prevalent RNA modification crucial for gene expression regulation. However, accurate and sensitive m5C sites identification remains challenging due to severe RNA degradation and reduced sequence complexity during bisulfite sequencing (BS-seq). Here, we report m5C-TAC-seq, a bisulfite-free approach combining TET-assisted m5C-to-f5C oxidation with selective chemical labeling, therefore enabling direct base-resolution m5C detection through pre-enrichment and C-to-T transitions at m5C sites. With m5C-TAC-seq, we comprehensively profiled the m5C methylomes in human and mouse cells, identifying a substantially larger number of confident m5C sites. Through perturbing potential m5C methyltransferases, we deciphered the responsible enzymes for most m5C sites, including the characterization of NSUN5's involvement in mRNA m5C deposition. Additionally, we characterized m5C dynamics during mESC differentiation. Notably, the mild reaction conditions and preservation of nucleotide composition in m5C-TAC-seq allow the m5C detection in chromatin-associated RNAs. The accurate and robust m5C-TAC-seq will advance research into m5C methylation functional investigation.

2024-07-11 | GSE242724 | GEO

Systematic Evaluation of Parameters in RNA Cytosine Methylation Analysis

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.

2022-04-05 | GSE190614 | GEO

rRNA 5-methylcytosine in Arabidopsis thaliana

Project description:Here we use bisulfite conversion of RNA combined with high-throughput IIlumina sequencing (RBS-seq) to identify single-nucleotide resolution of m5C sites in ribosomal RNAs of all three sub-cellular transcriptomes in Arabidopsis thaliana. m5C sites in rRNAs were also anlyzed in Arabidopsis T-DNA knockouts for the RNA methyltransferases TRM4A, TRM4B, TRDMT1, NSUN5, NOP2A, NOP2B and NOP2C.

2015-10-01 | GSE68444 | GEO

Bisulfite-sequencing of HBV 1.1 mer-transfected huh7 cells

Project description:RNA chemical modifications have been found to play important biological functions. Among which, the 5-methylcytosine (m5C) modification has been reported to participate in viral replication through affecting RNA processing, such as export, decay, translation and so on. In this study, we performed bisulfite sequencing (BS-seq) on HBV 1.1-mer-transfected huh7 cells to identify the m5C sites in HBV mRNA and their function in virus replication was verified. To investigate the mechanism by which m5C methyltransferase NSUN2 suppresses HBV replication, altered global m5C levels in host genes in HBV 1.1-mer-transfected cells were examined by BS-seq. We found that the m5C modification of genes associated with antiviral immunity changed significantly after viral infection. Our study provide new molecular insights into the mechanism of HBV-mediated IFN inhibition

2023-11-07 | GSE246879 | GEO

Transcriptome-wide RNA 5-methylcytosine methylation in siliques of Arabidopsis thaliana

Project description:Here we use bisulfite conversion of rRNA depleted RNA combined with high-throughput Illumina sequencing (RBS-seq) to identify single-nucleotide resolution of m5C sites transcriptome-wide in Arabidopsis thaliana siliques. m5C sites were also analyzed in an Arabidopsis T-DNA knockout for the RNA methyltransferase TRM4B.

2017-04-07 | GSE80050 | GEO

Epitranscriptomic maps of 5-methylcytosine reveal substrate diversity of NSUN methyltransferases and links to mRNA translation and turnover

Project description:Multiple epitranscriptomic maps of the RNA modification 5-methylcytosine (m5C) have been prepared, often diverging markedly from each other in terms of site abundance and identity. Differences in detection methods, data depth and analysis pipelines, but also biological factors underly much of this disparity. To address this, we re-analysed available datasets from five human cell lines and seven tissues, generated by the prevailing bisulfite RNA sequencing method, with a coherent m5C site calling pipeline. We used the resulting union list of 6,393 m5C sites called across the broader transcriptome to study site distribution, enzymology, interaction with RNA-binding proteins and molecular function. We confirmed prior observations such as predominance of m5C sites within mRNA exons, enrichment around start codons, and that the tRNA:m5C methyltransferases (MTases) NSUN2 and 6 are the main m5C ‘writers’ for mRNAs. Each NSUN enzyme recognises mRNA features that strongly resemble their respective canonical substrates. Assessing proximity between mRNA m5C sites and footprints of RNA-binding proteins reconfirmed the mRNA export factor ALYREF as an m5C ‘reader’ and identified new candidates for functional interactions, including the RNA helicases DDX3X, involved in mRNA translation, and UPF1, an mRNA decay factor. Experiments in HeLa cells lacking NSUN2 showed that this affected both, steady-state level and UPF1-binding to target mRNAs. Our studies emphasise the emerging diversity of both, m5C writers and readers, affecting mRNA function.

2024-07-04 | GSE252369 | GEO

Transcriptome-wide RNA 5-methylcytosine methylation in roots of Arabidopsis thaliana

Project description:Here we use bisulfite conversion of rRNA depleted RNA combined with high-throughput Illumina sequencing (RBS-seq) to identify single-nucleotide resolution of m5C sites transcriptome-wide in Arabidopsis thaliana roots. m5C sites were analyzed in wild type (WT) and an Arabidopsis T-DNA KO mutant for the RNA methyltransferase TRM4B.

2017-04-07 | GSE80052 | GEO

Ultrafast Bisulfite Sequencing for Efficient and Accurate 5-Methylcytosine Detection in DNA and RNA

Project description:Ultrafast Bisulfite Sequencing for Efficient and Accurate 5-Methylcytosine Detection in DNA and RNA

| PRJNA936787 | ENA