Expression profiling of METTL14 depleted human pancreatic β-like cells

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 explored the requirement of METTL14-mediated m6A RNA methylome in human pancreatic β-like cells by simultaneously inducing METTL14 knockdown in H1 human embryonic stem cells (hESCs) with doxycycline (Dox) treatment and differentiating hESCs towards pancreatic β-like cells in vitro. H1 hESCs were differentiated to Stage 6 (S6) β-like cells as reported previously (Kahraman et al. Nat Metab. 2022). Cells were harvested using TrypLE and neutralized in DMEM containing 10% FBS. The cell pellet was resuspended in DA-ZP1 containing cell media and incubated in 37°C for 30 min. Cells were washed with DPBS and the cell pellet was resuspended in fresh DA-ZP1–free media. Cells were sorted by Aria as described previously (Kahraman et al. Life Sci Alliance. 2021). Sorted cells were washed with DPBS and lysed in TRIzol for RNA isolation using RNeasy Micro Kit (Qiagen) according to the manufacturer's instructions. RNA libraries were prepared using SMARTer Stranded Total RNA-Seq kit v2 (Takara) and sequencing was performed on an Illumina NovaSeq 6000 according to the manufacturer instructions. Approximately 50 million paired-end 100-bp reads were generated for each sample.