Unknown,Transcriptomics,Genomics,Proteomics

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High resolution transcriptome-wide RNA cytosine methylome of Mouse Embryonic Fibroblasts


ABSTRACT: We report high resolution transcriptome-wide RNA cytosine methylome of Mouse Embryonic Fibroblasts (MEFs) revealed by an optimized new RNA bisulfite sequencing approach. Comparison of RNA methylation profile from wild-type and Dnmt2 -/- MEFs shows that only C38 in three tRNAs (tRNA-Asp, Gly and Val) is the target of Dnmt2 in MEFs at normal conditions. Harvested MEFs, from 13.5 isogenic (wt or Dnmt2-/-) embryos, were subjected to total RNA isolation and DNase treatment. Small RNA fraction was separated using mirVana kit (Ambion). Large RNA fraction was prepared by ribosomal RNA depletion using RiboMinus™ Transcriptome Isolation Kit (Invitrogen) followed by RNA fragmentation using RNA Fragmentation Reagent (Ambion). Each one of the fractions (small or large) from each one of the samples (wt or Dnmt2-/-) were split into two: one for direct RNA sequencing and one for RNA bisulfite sequencing. For bisulfite treatment, small and large RNA fractions of each sample (wt or Dnmt2-/-) were separately subjected to bisulfite treatment. Total of 8 samples (bisulfite treated and untreated of small and large RNA fractions of wt or Dnmt2-/-) were separately subjected to library preparation with Illumina’s directional mRNA-Seq sample preparation protocol followed by 101 cycle single-end high-throughput sequencing using Illumina’s HiSeq 2000 sequencing system.

ORGANISM(S): Mus musculus

SUBMITTER: Vahid Khoddami 

PROVIDER: E-GEOD-44359 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Identification of direct targets and modified bases of RNA cytosine methyltransferases.

Khoddami Vahid V   Cairns Bradley R BR  

Nature biotechnology 20130421 5


The extent and biological impact of RNA cytosine methylation are poorly understood, in part owing to limitations of current techniques for determining the targets of RNA methyltransferases. Here we describe 5-azacytidine-mediated RNA immunoprecipitation (Aza-IP), a technique that exploits the covalent bond formed between an RNA methyltransferase and the cytidine analog 5-azacytidine to recover RNA targets by immunoprecipitation. Targets are subsequently identified by high-throughput sequencing.  ...[more]

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