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M6a peak analysis in normal, FTO deficient and five stages of adipogenesis (D-2/0/2/5/10) in Mouse embryo fibroblast 3T3-L1 pre-adipocytes

ABSTRACT: Total RNA was extracted using TRI® Reagent (Sigma). cDNA was synthesized by RevertAid™ First Strand cDNA Synthesis Kit with Oligo dT primers (K1622, Fermentas) following manufacturer’s recommendations. PCR reactions were carried out on a DNAEngine® Thermal Cycler (PTC-0200G, Bio-Rad) in 25 μl reaction volume containing 1 μl cDNA, 200 nM primer pairs and components of TaKaRa Taq™ kit (R001A, Takara). All samples were analyzed in triplicate RT-qPCR.mRNAs were extracted using biotinylated poly(dT) oligo, followed by further removing of contaminated rRNA using RiboMinus Transcriptome Isolation Kit (K1550-02, Invitrogen). Then mRNAs were fragmented into 100-200nt length and subjected to immunoprecipitation with m6A specific antibody.The libraries were sequenced using HiSeq2000 (Illumina) in single-read mode, creating reads with a length of 101 bp. Sequencing chemistry v2 (Illumina) was used and samples were multiplexed in two samples per lane.

ORGANISM(S): Mus musculus

PROVIDER: GSE53244 | GEO | 2015/03/09

SECONDARY ACCESSION(S): PRJNA231244

REPOSITORIES: GEO

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m6a peak analysis in normal, FTO deficient and five stages of adipogenesis (D-2/0/2/5/10) in Mouse embryo fibroblast 3T3-L1 pre-adipocytes

Project description:Total RNA was extracted using TRI® Reagent (Sigma). cDNA was synthesized by RevertAid™ First Strand cDNA Synthesis Kit with Oligo dT primers (K1622, Fermentas) following manufacturer’s recommendations. PCR reactions were carried out on a DNAEngine® Thermal Cycler (PTC-0200G, Bio-Rad) in 25 μl reaction volume containing 1 μl cDNA, 200 nM primer pairs and components of TaKaRa Taq™ kit (R001A, Takara). All samples were analyzed in triplicate RT-qPCR.mRNAs were extracted using biotinylated poly(dT) oligo, followed by further removing of contaminated rRNA using RiboMinus Transcriptome Isolation Kit (K1550-02, Invitrogen). Then mRNAs were fragmented into 100-200nt length and subjected to immunoprecipitation with m6A specific antibody.The libraries were sequenced using HiSeq2000 (Illumina) in single-read mode, creating reads with a length of 101 bp. Sequencing chemistry v2 (Illumina) was used and samples were multiplexed in two samples per lane. discovery of the binding motif of m6a in normal, FTO deficient and five stages of adipogensis (D-2/0/2/5/10) in Mouse embryo ﬁbroblast 3T3-L1 pre-adipocytes

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