Project description:We report the application of m6A-RIP-seq to indentify the m6A modifications variation in genes invovled in EMT. Briefly, total polyadenylated RNA was isolated from HeLa cells treated with or without 10 ng/ml TGF-β for 3 days by use of TRIZOL reagent followed the using of FastTrack MAGMaxi mRNA isolation kit (Invitrogen). RNA fragmentation, m6A-seq, and library preparation were performed according to instructions of manufacture and the previously published protocol (Wang et al., 2015). NEBNext Ultra Directional RNA Library Prep Kit (New England BioLabs, Ipswich, MA) was used for library preparation. Each experiment was conducted in two biological replicates. m6A-seq data were analyzed according to the protocols described before (Wang et al., 2015). Significant peaks with FDR < 0.05 were annotated to RefSeq database (hg19). Sequence motifs were identified by using Homer. Gene expression was calculated by Cufflinks using the sequencing reads from input samples. Cuffdiff was used to find the DE genes.
Project description:To investigate the effect of HSATIII lncRNA on m6A modification, we performed m6A-RIP(RNA immuno precipitation) RNA-seq from heat shock-exposed HeLa cells upon HSATIII knockdown.
Project description:N6-methyladenosine (m6A) is a widespread reversible chemical modification of RNAs, implicated in many aspects of RNA metabolism. Little quantitative information exists as to either how many transcript copies of particular genes are m6A modified (âm6A levelsâ), or the relationship of m6A modification(s) to alternative RNA isoforms. To deconvolute the m6A epitranscriptome, we developed m6A level and isoform-characterization sequencing (m6A-LAIC-seq). We found that cells exhibit a broad range of non-stoichiometric m6A levels with cell type specificity. At the level of isoform characterization, we discovered widespread differences in use of tandem alternative polyadenylation (APA) sites by methylated and nonmethylated transcript isoforms of individual genes. Strikingly, there is a strong bias for methylated transcripts to be coupled with proximal APA sites, resulting in shortened 3â untranslated regions (3â-UTRs), while nonmethylated transcript isoforms tend to use distal APA sites. m6A-LAIC-seq yields a new perspective on transcriptome complexity and links APA usage to m6A modifications. m6A-LAIC-seq of H1-ESC and GM12878 cell lines, each cell line has two replicates
Project description:N6-methyladenosine (m6A) is a widespread internal RNA modification whose function is poorly understood. Here we report that m6A residues within the 5'UTR promote a novel form of cap-independent translation which is mediated through an interaction between m6A residues and the translation initiation factor, eIF3. We present eIF3a PAR-iCLIP data which demonstrate that eIF3 predominantly binds mRNAs within the 5'UTR. eIF3 binding sites are also in proximity to m6A residues within the 5'UTR of cellular mRNAs. Two replicates of eIF3a PAR-iCLIP in HEK293T cells.
Project description:We report the application of MeRIP-seq to map m6A peaks in wild type and METTL5 KO HeLa cells to investigate targets of the m6A methyltransferase METTL5.
Project description:MeRIPSeq of HeLa cells synchronized by a double thymidine block to obtain the cell cycle phases and work with total RNA to study m6A mark in mRNA without polyA tail RNA seq of HeLa cells synchronized by a double thymidine block to obtain the cell cycle phases and work with total RNA to study mRNA without polyA tail
