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:Methylation individual-nucleotide-resolution crosslinking and immunoprecipitation (miCLIP) was performed in human primary CD8⁺ T cells subjected to in vitro activation with αCD3/αCD28 beads. To map m⁶A sites and assess their dynamic signatures during T cell activation, we applied an improved iCLIP (iiCLIP) protocol using an anti-m⁶A antibody (Abcam) on 750–100 ng of polyadenylated RNA. Samples were collected from non-activated (NoAct), Day 1-activated, and Day 5-activated CD8⁺ T cells isolated from healthy donors (NoAct: 4 donors; Day 1: 5 donors; Day 5: 4 donors). Two experimental controls were included: (i) “Input” libraries prepared without the m⁶A immunoprecipitation step (4 donors each for NoAct, Day 1, and Day 5), and (ii) “noUV” libraries generated without UV crosslinking (3 donors total).
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.
