GSM8005230: RVSMC OE Mettl3 IP2; Rattus norvegicus; RIP-Seq
MeRIP experiments, high-throughput sequencing and data analysis were conducted by Seqhealth Technology Co., Ltd. (Wuhan, China). Briefly, total RNA was extracted from control or Mettl3-overexpressing rat primary VSMCs using TRIzol Reagent (Invitrogen, cat. No. 15596026). One hundred micrograms of total RNA were used for the meRIP experiment. Briefly, 20 mM ZnCl2 was added to total RNA and incubated at 94°C for 5 min until the RNA fragments were mainly distributed in 100-200 nt. Then, 10% of the RNA fragments were saved as “input”, and the remaining fragments were used for m6A immunoprecipitation (IP). The specific anti-m6A antibody (Synaptic Systems, 202003) was applied for m6A IP. The stranded RNA sequencing library was constructed by using the KC-DigitalTM Stranded mRNA Library Prep Kit for Illumina® (Catalog No. DR08502, Wuhan Seqhealth Co., Ltd., China) with five PCR cycles. The kit eliminates duplication bias in PCR and sequencing steps. Then, ribosomal cDNA was removed by a SMARTer Stranded Total RNA-Seq Kit version 2 (Pico Input Mammalian; 634413; TaKaRa/Clontech, Japan), and ten PCR cycles were added. The library products corresponding to 200-500 bp were enriched, quantified and finally sequenced on a DNBSEQ-T7 sequencer (MGI Tech Co., Ltd., China) with a PE150 model.