N6-methyladenosine profiling of differentiating human pancreatic β-cells in vitro

N6-methyladenosine (m6A) is the most abundant chemical modification in mRNA, and plays important roles in human embryonic stem cell pluripotency, maintenance, and differentiation. However, the role of m6A and the precise mechanisms involved during the development of β-cells are unexplored. Here, we differentiated human embryonic stem cells (hESCs) into pancreatic β-like cells and performed RNA-Seq and m6A-seq at different stages of in vitro β-like cell differentiation. H1 and MEL1 hESCs were differentiated to Stage 3 (S3), Stage 4 (S4) and Stage 6 (S6) as reported previously (Kahraman et al. Nat Metab. 2022). Total RNA was isolated by TRIzol reagent and mRNAs were purified by Dynabeads mRNA purification kit. Purified mRNA was fragmented by Bioruptor® Pico Sonication System and input was saved before m6A immunoprecipitation. m6A immunoprecipitation was performed with EpiMark®N6-Methyladenosine Enrichment Kit (NEB, E1610S) following the manufacturer protocol. Then, RNA libraries were prepared for both input and IP samples using TruSeq® Stranded mRNA Library Prep (Illumina, 20020594) following the manufacturer protocol. Sequencing was performed at the University of Chicago Genomics Facility on an Illumina NovaSeq 6000 machine.