Expression profiling of METTL14 depleted human embryoid bodies
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 ß-cells are unexplored. Here, we explored the requirement of METTL14-mediated m6A RNA methylome in human embryonic stem cell (hESC) differentiation by simultaneously inducing METTL14 knockdown in H1 and MEL1 hESCs with doxycycline (Dox) treatment and differentiating hESCs towards the three embryonic germ layers using embryoid body (EB) formation assays. Overall design: Inducible METTL14 KD H1 and MEL1 hESCs were generated by transducing the cells with SMARTvector human lentiviral vectors containing shRNAs targeting METTL14 from Dharmacon (iKD1; V3SH7669-224822773, iKD2; V3SH7669-225341368, iKD3; V3SH7669-229883026, iSCR; VSC10712). Puromycin (2 µg/ml) was added to the culture media starting from post transduction day 4 to select stable inducible knockdown hESCs. Inducible METTL14 KD H1 and MEL1 hESCs were dissociated into single cells and 500 cells per microwell were seeded in AggreWell™400 24 well plates according to the manufacturer instructions. AggreWell™ EB Formation Medium - supplemented with doxycycline (2µg/ml) to induce shRNA expression - was refreshed every other day for ten days. EBs were then harvested, washed with DPBS, and lysed in TRIzol for RNA isolation using RNeasy Kit (Qiagen) according to the manufacturer instructions. RNA libraries were prepared using TruSeq Stranded Total Library Prep (Illumina) and sequencing was performed on NovaSeq 6000 according to the manufacturer instructions. Approximately 50 million paired-end 100-bp reads were generated for each sample.