The Landscape of N-6 Methyladenosine in Primary Localized Prostate Cancer
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ABSTRACT:
PROVIDER: EGAS00001006925 | EGA |
REPOSITORIES: EGA
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Project description:Using meRIP-sequencing, we profiled N-6 methyladenosine (m6A) in a cohort of 148 primary prostate cancer samples as part of the Canadian Prostate Cancer Genome Network project (CPC-GENE). Paired-end sequencing of 150 bp reads were mapped to GRCh38.p13 using annotations from gencode.v34.chr_patch_hapl_scaff.annotation.gtf. Peaks were called using MeTPeak, joint peaks were identified and IP reads were quantitated, normalized and adjusted using Input reads to obtain estimates of m6A abundance.
| EGAD00001009869 | EGA
Project description:m6A RNA immunoprecipitation was performed according to published procedure (Dominissini et al., 2012). Human m6A-seq data from DTSC2 HeLa cells were aligned to the hg19 transcriptome. To locate m6A peaks, the hg19 transcriptome was divided into 25 nucleotide-wide tiles. The number of reads in the m6A immunoprecipitation (IP) and non-IP (control) sample was counted in each tile, and a p-value was calculated using Fisher’s exact test and adjusted for multiple testing. Tiles with significant enrichment of the m6A signal (adjust-P value < = 0.05) were merged into larger regions. Regions lesser than 100 bp were discarded, and regions over 200 bp were separated into 100 to 200 bp sub-regions; the m6A signal from rapamycin-treated cells compared to control (DMSO) was calculated for each region; and regions with at least a two-fold enrichment in all replicates were identified as m6A peaks. The distributions of m6A peaks and m6A marked genes were identified by overlapping all m6A peaks with the hg19 RefGene annotation (Meng et al., 2014).
2021-02-17 | GSE165690 | GEO
Project description:The goal of the experiment is to determine the m6A tagged transcripts after the Mettl14 gene inactivation in OPCs and mature oligodendrocytes using Z score > 0 . Methods: mRNA from total RNA of Mettl14fl/fl OPCs and mature oligodendrocytes was purified with Dynabeads Oligo (dT)25 (ThermoFisher; 61006). The equivalent amount of input and m6A-imunoprecipitated RNA were prepared for library generation using the SMART-seq protocol as described (Picelli et al., 2014). Three biological replicates of control OPCs and three biological replicates of control oligodendrocytes were sequenced using Illumina NextSeq 500. Adapters were trimmed from original reads using Trimmomatic and low-quality reads were removed. The remaining reads were then mapped to the mouse genome (mm10) using STAR aligner (Dobin et al., 2013). To measure the relative m6A level per gene, the ratio of m6A IP/ Input was first calculated. The Z scores were then obtained by comparing the ratios (m6A IP/ Input) to the mean of the group to reflect the relative m6A level per gene on a transcriptome-wide scale. Results: The m6A-seq analyses detected 3554 m6A marked transcripts in OPCs and 2606 m6A marked transcripts in oligodendrocytes.There are 2806 transcripts with the m6A mark in OPCs that were present but not marked in oligodendrocytes , and 1626 transcripts that possessed the m6A mark in oligodendrocytes but not in OPCs . Only 23 of the shared transcripts , showed the m6A mark in both OPCs and oligodendrocytes. Conclusions: The dynamic nature of the m6A mark in oligodendrocyte lineage cells suggests that it may play an important role in oligodendrocyte differentiation and CNS myelination.
2019-12-31 | GSE124202 | GEO
Project description:Total RNAs were isolated from WT splenic NK cells, and subjected to standard m6A MeRIP, in two replicates, using Illumina Novaseq 6000 platform. The raw sequencing reads were mapped to the genome of Mus musculus (mm10) with default parameters. ExomePeak was used to identify m6A peaks, which were annotated by intersection with gene architecture using ChIPseeker. Sequence motifs enriched in peak regions were identified using Homer.
2021-07-20 | GSE173326 | GEO
Project description:ChIP Seq revealed presence of unique CEN-proximal neocentromere peaks in CEN deleted clones. Using 10 million reads generated from ChIP-seq for each sample, chromosome wide maps for CENP-A enrichment were generated . Examination of CENP-A enrichment in Input/IP samples of various CEN deleted C. albicans clones
2012-12-21 | E-GEOD-42907 | biostudies-arrayexpress
Project description:We report the application of MeRIP sequencing technology for high-throughput profiling of RNA m6A modifications in wide-type and knock-down METTL3 or WTAP Human Umbilical Vein Endothelial Cells (HUVECs). Both the input samples without immunoprecipitation and the m6A IP samples were used for RNA-seq library generation with NEBNext® Ultra II Directional RNA Library Prep Kit (New England Biolabs, Inc., USA). Library sequencing was performed on an illumina Hiseq instrument with 150bp paired-end reads. Clean reads of all libraries were aligned to the reference genome (HG19) by Hisat2 software (v2.0.4). Methylated sites on RNAs (peaks) were identified by MACS software. Differentially methylated sites were identified by diffReps. And, guided by the Ensembl gtf gene annotation file, cuffdiff software (part of cufflinks) was used to get the gene level FPKM as the expression profiles of mRNA, and fold change and p-value were calculated based on FPKM, differentially expressed mRNA were identified. qRT-PCR validation was performed using SYBR Green assays. Finally, we find that METTL3/WTAP can regulate the expression level of target genes through m6A modification in HUVECs. This study provides a framework for applying MeRIP sequencing profiles to characterize vascular endothelial cells.
2019-12-20 | GSE142386 | GEO
Project description:We performed m6A-RIPs in Ascl1-induced neurons (iNeurons) to investigate the neuronal m6A epitranscriptome. Immunoprecipitation was done twice using two different antibodies, acquired from Abcam and Synaptic Systems (SySy), allowing for a more robust detection of m6A modification marks. Additionally, RIP-seq was performed separately with intact and fragmented RNA. The former approach allowed to identify proportions of m6A-modified transcripts among the total number, while the latter approach provided the information to identify genomic coordinates of m6A peaks.
2023-07-12 | E-MTAB-9542 | biostudies-arrayexpress
Project description:N6-methyladenosine (m6A) is one of the most abundant modifications in eukaryotic RNA. Recent mapping of m6A methylomes in mammals, yeast, and plants as well as characterization of m6A methyltransferases, demethylases, and binding proteins have revealed regulatory functions of this dynamic RNA modification. In bacteria, although m6A is present in ribosomal RNA (rRNA), its occurrence in messenger RNA (mRNA) still remains elusive. Here, we used liquid chromatography-mass spectrometry (LC-MS) to calculate the m6A/A ratio in mRNA from a wide range of bacterial species, which demonstrates that m6A is an abundant mRNA modification in tested bacteria. Subsequent transcriptome-wide m6A profiling in Escherichia coli and Pseudomonas aeruginosa revealed a conserved distinct m6A pattern that is significantly different from that in eukaryotes. Most m6A peaks are located inside open reading frames (ORF), and carry a unique consensus motif (GCCAU). Functional enrichment analysis of bacterial m6A peaks indicates that the majority of m6A-modified transcripts are associated with respiration, amino acids metabolism, stress response, and small RNAs genes, suggesting potential regulatory roles of m6A in these pathways. m6A profiling in E.coli and P.aeruginosa mRNA
2015-07-08 | E-GEOD-65347 | biostudies-arrayexpress
Project description:To investigate the dynamic m6A modification during starvation-induced autophagy, we performed MeRIP-seq in MEFs with or without nutrient deprivation and identified the variations of m6A modification on mRNAs. Briefly, the total polyadenylated mRNAs in MEFs cultured with or without nutrient-deprived medium for 2 hours were isolated using RNAiso plus reagent (Takara) followed Dynabeads mRNA Purification Kit (Invitrogen). The m6A RNA immunoprecipitation was performed using GenSeq m6A RNA IP Kit. NEBNext Ultra II Directional RNA Library Prep Kit (NEB) was used for library generation. Each experiment was conducted in two biological replicates. Methylated sites on peaks were identified by MACS2 peak-calling software (Zhang et al., 2008). Differentially methylated sites were identified by diffReps (Shen et al., 2013) with default setting.
2021-12-30 | GSE158268 | GEO
Project description:Prostate cancer gene expression profiles were studied in this project. A total RNA from 148 prostate sample with various amount of different cell types were hybridized to Affymetrix U133A arrays. The percentage of different cell types vary considerably among samples and were determined by pathologist. Cell type specific genes can be determined by linear regression using the methods of Stuart et al, PNAS, 2004. Experiment Overall Design: 148 prostate samples, with various amounts of tumor, stroma, BPH and atrophic gland, were used for this study.
2008-06-16 | E-GEOD-8218 | biostudies-arrayexpress