Polyttract mrna isolation system

Total RNA was extracted and purified by PolyTtract mRNA Isolation System (Promega). After fragmentation, magna methylated RNA immunoprecipitation m6A Kit (Merck Millipore, Germany) was used for m6A RNA immunoprecipitation. In short, total RNA was dispersed into 100nt-long oligonucleotides and then incubated with anti-m6A antibodies or immunoglobulin (IgG). The fragmented RNA containing m6A was eluted and purified with RNA purification kit (Qiagen, Germany). Enriched fragments were analyzed by qPCR. The purified RNA fragments were used to construct libraries using the NEBNext Ultra RNA Library Prep Kit (New England Biolabs, USA) and sequenced using Illumina HiSeq 2000 (Illumina, USA) platform. The level of m6A mRNA was measured by qPCR.

Total RNAs were extracted and purified by using PolyTtract mRNA Isolation System (Promega, Hong Kong). After fragmentation, RNA was incubated with m⁶A antibody for immunoprecipitation according to the standard protocol of the Magna methylated RNA immunoprecipitation m⁶A Kit (Merck Millipore, Germany). Enrichment of m⁶A containing mRNA was then analyzed either through RT-qPCR or high-throughput sequencing. Primers to m⁶A negative region of EEF1A was used as the negative control and primers to m⁶A postive region of EEF1A was used as the positive control according to the standard protocol of the Magna methylated RNA immunoprecipitation m⁶A Kit (Merck Millipore, Germany). For high-throughput sequencing, purified RNA fragments were used for library construction with the NEBNext Ultra RNA library Prep kit for Illumina (New England BioLabs) and were sequenced with Illumina HiSeq X Ten platform. Library preparation and high-throughput sequencing were performed by Novogene (Beijing, China).