GSM5775613: MDAMB231_m6A-seq_shNS_IP_rep2; Homo sapiens; OTHER
Trizol (Qiagen) was used to isolate total RNA followed by PolyA RNA purification using PolyATtract® mRNA Isolation Systems kit (Promega) from MDA-MB-231 cells. The RNA was fragmented by incubating mRNA at a concentration of 1ug/ul with 10mM ZnCl2 in 10mM Tris-HCl pH 7.0 at 94C for 4 minutes. 10ug of fragmented RNA was then immunoprecipitated using 5ug anti-m6A antibody (Synaptics Systems). The immunoprecipitated RNA was purified by the RNeasy MinElute Cleanup Kit (Qiagen). Agilent Bioanalyzer 2100 was used to determine the size and quality of both IP and input samples prior to RNA library construction using KAPA Stranded mRNA-Seq Kit (Illumina Platforms) (KR0960m Kapa Biosystems) and sequenced on an Illumina Hiseq2500.