GSM6256887: ligamentum flavum ,non ossified ,rep 3; Homo sapiens; OTHER
Trizol reagent (Invitrogen) was applied to extract total RNA. MeRIP-Seq was performed by Cloudseq Biotech Inc. (Shanghai, China) according to the published procedure with proper adjustments. Briefly, the fragmented RNA was incubated with m6A polyclonal antibody (Synaptic Systems, 202003) in IPP buffer at 4°C for 2 hours. Then the mixture was immune-precipitated by incubation with protein-A beads (Thermo Fisher) at 4°C for an additional 2 hours. Then, bound RNA was eluted from the beads with N6-methyladenosine (BERRY & ASSOCIATES, PR3732) in IPP buffer and then extracted with Trizol reagent (Thermo Fisher) following the manufacturer's instruction. Purified RNA was used for RNA-seq library generation with NEBNext® UltraTM RNA Library Prep Kit (NEB). Both the input sample without immunoprecipitation and the m6A IP samples were subjected to 150 bp paired-end sequencing on Illumina HiSeq sequencer. In addition, the q-PCR was performed to detect the level of m6A using purified RNA fragments