Project description:GP61-primed effector CD4+ T cells were isolated from Ctrl or Mettl3-deficient SMARTA mice. Total RNAs were extracted with TRIzol reagent, and mRNAs were then isolated with Dynabeads® mRNA purification kit, followed by stardard m6A-miCLIP-SMARTer-seq with some modifications. Raw sequencing reads were aligned to the mouse genome (mm10) with BWA, and then m6A sites were determined.
Project description:To understand the global effect of H3K36me3 on m6A modification, we compared the m6A profiling in SETD2 knockdown and control HepG2 cells by miCLIP-seq, and found the depletion of H3K36me3 by SETD2 silencing globally reduced m6A in the human transcriptome.
Project description:To identify m6A sites on endogenous nuclear RNAs, we performed miCLIP to identify m6A sites in PANC-1 cells. To identify NKAP binding sites on endogenous nuclear RNAs, we performed iCLIP for flag-tag NKAP to analyze the nuclear RNA binding with NKAP in the same cells.
Project description:RNA-sequencing was performed on the following human neuroblastoma cell lines: Kelly, NBL-S, CHP-212, SH-SY5Y, SH-SY5Y LDK-resistant and SH-EP.
Project description:We employed miCLIP-seq to profile the location and extent of m6A in the Poly(A)+ transcriptome. Skin epithelial cells were isolated from wild-type P0 neonates by FACS. Total RNA was extracted by TRIzol-LS and Poly(A)+ RNA was extracted with Dynabeads™ mRNA Purification Kit (Thermo-Fisher, 61006). Input and miCLIP libraries was prepared from 3 biological replicates with each containing RNA isolated from 3 litters of neonates. Libraries were sequenced on the Illumina Hi-seq platform to generate paired-ended 50 bp reads. Sequencing data was processed as described in (Geula et al., 2015).
Project description:Aberrant post-transcriptional methylation is implicated in a wide range of human diseases, yet the specific enzymes responsible for site- and substrate-specific methylation remain largely unknown. Here, we use our recently developed catalysis-dependent RIP sequencing approach (miCLIP) and RNA bisulfite sequencing to map C5-methylcytosine (m5C) in the human transcriptome. We identified two novel m5C methylases NSun3 and NSun6, both of which have strong substrate specificity. In contrast to the previously characterized NSun2, which displays some preference to methylating transfer RNAs (tRNA), NSun6 predominantly targeted 3’ UTRs of messenger RNAs (mRNA), and NSun3 mainly methylated mitochondrial RNAs (mtRNA). Consistent with the miCLIP-predicted RNA target specificity, whole exome sequencing identified NSUN3 loss-of-function mutations in a patient presenting with combined respiratory chain complex deficiency. Functional studies of the patient fibroblast cell line revealed that loss of the NSun3 protein resulted in severe mitochondrial translation defects, which were rescued by expression of wild-type NSun3. In summary, our results reveal how highly conserved NSun m5C methylases partition their substrate specificities to remodel the sequences of particular ribonucleotide classes.
Project description:ChIP-seq to define bindings sites for TBX2, MYCN, H3K27ac, H3K4me1, H3K4me3 and Input in the cell lines IMR-32, CLB-GA, NGP, IMR-5/75, GI-M-EN, CHP-212, and IMR-5.
