MeRIP-seq to identify putative Mettl3 targets in the nephron progenitor cells

Mettl3 is an mRNA methyl transferase enzyme. It catalzyes the methylation of the most common mRNA biochemical modification N6-methyl adenosine (m6A). The role of Mettl3/m6A pathway is unknown in the developing kidney. Through genetic and pharmaceutical approaches we found that Mettl3 is required for the differentiation of nephron progenitor cells. nephron progenitors. Genetic deletion of Mettl3 in the nephron progenitor cells blocks their differentiation. We sought to identify the mechanism of Mettl3 activity. We performed RNA-sequencing and RNA-m6A pull down assay followed by next-generation sequencing (MeRIP-seq) on wild type and Mettl3 deleted kidneys to determine the mRNA targets of Mettl3. Overall design: m6A RNA immunoprecipitation was performed using the m6A antibody precipitation approach. RNA isolated from kidneys of Six2Cre or Six2Cre-Mettl3-KO E16.5 pups was subjected to fragmentation to obtain fragments of 100 nucleotides or smaller in length. Magnetic immunoprecipitation was performed. Two biological replicate samples per group were analyzed, each sample containing pooled fragmented RNA from 4-6 pairs of kidneys. The samples were subjected to Illumina Illumina's next-generation sequencing.