Project description:We have performed a epitranscriptomics study in which we first tretaed TNF-alpha in HeLa cells and DMSO was used as a negative control. Total RNA was isolated from TNF-alpha treated cells as well as control cells and apoptotic rate was determined by flow cytometry. Total RNAs were subjected to miCLIP to identify differentially m6A methylated RNAs. We then transcriptionally analyzed apoptotic genes which have differential m6A methylation in TNF-alpha tretament by qPCR. Afterthat, candidate genes were examined at the level of translation by polysome fractioantion assay.
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: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
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.
Project description:Toxoplasma gondii (T. gondii) is an opportunistic parasite. After infection, macrophages finely regulate the immune response to restrict parasite proliferation. It is well-known that N6-methyladenosine (m6A) plays a critical role in fine-tuning gene expression. To investigate whether m6A modification is involved in regulating the anti-infection immune response in macrophages against T. gondii, this study utilized T. gondii tachyzoites from the RH strain to infect human THP-1 macrophages. qPCR and ELISA results showed that T. gondii infection mounted the expression of TNF-α. RNA-seq profiling showed that T. gondii infection was associated with difference in genes from pathway associated with TNF signaling. Expression of m6A regulators were evaluated using qPCR and Western blotting. T. gondii infection increased the abundance of m6A methyltransferase WTAP and demethylase FTO. Joint analysis of RNA-seq and m6A-seq data was utilized for enriching differentially expressed genes with significantly altered m6A modifications. After T. gondii infection, the m6A levels of genes associated with TNF signaling were significantly altered. In this study, we found that m6A methylation involved in T. gondii infection induced TNF-α expression.