Anti m6a

For RNA immunoprecipitation (RIP), two rounds using 5 μg of anti-m6A (#ab208577, Abcam, Paris, France), anti-m7G (#6655, Biovison, Milpitas, CA, USA), and anti-m5C (#61255, Active Motif, Nantes, France) antibodies and 5 μg of small RNA were performed. The reaction was carried out using a Dynabeads protein G IP kit with some modifications (#10007D, Thermo Fisher Scientific, Paris, France) such as described by Berulava et al. (2015) [14 (link)] and Briand et al. (2021) [19 (link)]. As a control, IP was performed using IgG (#ab18443, Abcam, Paris, France) instead of an anti-m6A antibody. miRNAs obtained from RIP were reverse transcribed using a miScript II RT kit (#218161, Qiagen, Paris, France) and analyzed using the miScript miRNA PCR array human cancer pathway kit (#331221, Qiagen, Paris, France) according to the manufacturer’s instructions. Fold enrichment was next calculated using the Ct value obtained from qRT-PCR performed with input miR, IP-IgG, IP-m6A, and the 2^−ΔΔCt formula.

MeRIP-qPCR was performed to quantify the m⁶A-modified levels of TROAP. Specifically, total cellular RNA was isolated from SKOV-3 cells using Trizol regent. The RNA samples (100 μg for each reaction) were incubated with 3 μg anti-m⁶A (Cat. #208577; Abcam, Cambridge, MA, USA) or anti-IgG (Cat. #172730; Abcam) that were conjugated with A/G magnetic beads in an immunoprecipitation buffer containing RNase inhibitor and protease inhibitors. Next, RNA from the mixtures was collected through centrifugation, cleaned using a phenol-chloroform solution, and eluted with elution buffer. The RNA samples (10 ng for each reaction) were then subjected to reverse transcription using Superscript III random hexamers (Invitrogen, Shanghai, China), and the cDNA was further analyzed by qPCR. The TROAP primer sequences that were used for MeRIP-qPCR were 5′-TTGCGGCGTCTCACCGTTCAACCT-3′ and 5′-GCCTCCATTAAGAGGGACACACTGG-3′. TROAP mRNA levels were quantified using the 2^-ΔΔCt associated sample addition method for fold enrichment.