Specific hsa mir miscript primer assays

RNA from cells was extracted with the NucleoSpin RNA kit (Macherey-Nagel, Hoerdt, France). RNA from tumors was extracted with Maxwell RSC simplyRNA Tissue Kit (Promega). Reverse transcriptions of 500 ng of RNA were performed with PrimeScript RT-PCR kit (Takara, Ozyme, France). Quantitiative polymerase chain reaction (qPCR) were performed on the CFX Connect Real Time System (Bio-Rad, France) using TB Green® Premix Ex Taq™ (Takara, France), following manufacturer’s instructions. Quantity of mRNA was analyzed using the ΔΔCT method normalized to the housekeeping genes HPRT and ALAS-1 for cell experiments and HPRT and TBP for tissue experiments. Primers were purchased at Eurogentec (Seraing, Belgium), and their sequences are detailed in Supplemental Table S7. The miRNA were extracted with miRNEasy mini kit (Qiagen, Courtaboeuf, France) and were reverse transcribed using the miRScript II RT kit (Qiagen) and analyzed by qPCR with the miScript SYBR Green PCR Kit (Qiagen) using the specific hsa-miR miScript Primer Assays (Qiagen) according to the manufacturer's instructions. miRs expression fold changes were calculated using the 2-ΔΔCt formula and SNORD61 and RNU6-2 as normalizers according to the manufacturer’s instructions.

For miRNA expression analysis and detection from products of RIP performed with anti-m6A antibody, RNA was reverse transcribed using a miScript II RT kit and analyzed by qPCR with the miScript SYBR Green PCR kit using the specific hsa-miR miScript primer assays (QIAGEN, France) according to the manufacturer’s instructions.

For miRNA expression analysis, the RNA was reverse transcribed using a miRScript II RT kit and analyzed by qPCR with the miScript SYBR Green PCR Kit using the specific hsa-miR miScript Primer Assays (Qiagen, France) according to the manufacturers’ instructions. miRs expression fold changes were calculated using the ^2-ΔΔCt formula and SNORD61 as normalizer according to the manufacturer’s instructions.