title: GSM8943082: MIA PaCa-2 si NC-1 input; Homo sapiens; RIP-Seq
library_construction_protocol: To determine the m6A levels of c-Myc mRNA, total RNA was extracted and fragmented using NEBNext® Magnesium RNA Fragmentation Module (NEB; Ipswich, MA, USA), followed by cleaning with Monarch® RNA Cleanup Kits (NEB). Then, short RNA fragments were removed and mRNA was fragmented into approximately 200 nt nucleotides. Methylated RNA immunoprecipitation (MeRIP) was performed according to the instructions of the EpiMark® N6-Methyladenosine Enrichment Kit (NEB). The co-precipitated RNA was converted to cDNA for sequencing library construction and sequenced on Illumina NovaSeq 6000 (Novogene; Beijing, China). Library preparation: The mRNA m6A was sequenced by MeRIP-seq at Novogene (Beijing, China). Briefly, a total of 30 µg RNAs were extracted from treated cells. The integrity and concentration of extracted RNAs were detected using an Agilent 2100 bioanalyzer (Agilent) and simpliNano spectrophotometer (GE Healthcare), respectively. Fragmented mRNA (~100 nt) was incubated for 2 hr at 4℃ with antim6A polyclonal antibody (Synaptic Systems) in the immunoprecipitation experiment. Then, immunoprecipitated mRNAs or Input was used for library construction with NEB Next® Ultra™ RNA Library Prep Kit (New England Biolabs). The library preparations were sequenced on an Illumina Novaseq platform with a paired-end read length of 150 bp according to the standard protocols.