GSM8757779: KO3_ip; Mus musculus; RIP-Seq
The Nano UMI MeRIP experiment, high-throughput sequencing and data analysis were conducted by Seqhealth Technology Co., Ltd. (Wuhan, China). The right ventricle and outflow tract tissues from E10.5-11.5 Mettl3-CV KO and control embryos were isolated. Each group consisted of two biological duplicate samples and each sample contained tissues from 20 embryos. The RNA was extracted using TRIzol reagent. Total RNA (5 μg) was fragmented into approximately 200 nt fragments and used for the MeRIP experiment. A specific anti-m6A antibody (Synaptic Systems, 202003) was used for m6A immunoprecipitation. 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 by using a unique molecular identifier (UMI) of 8 random bases to label the preamplified cDNA molecules. Then, ribosomal cDNA was removed with 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 of 200-500 bp were enriched, quantified and finally sequenced on a PE150 model DNBSEQ-T7 sequencer (MGI Tech Co., Ltd., China).