GSM2719428: Flag; Homo sapiens; OTHER
Dissociated cells are UV crosslinked on ice for 400 mJ/cm2, leading to formation of a covalent bond between protein and RNA. After lysis, the ligated RNA product was treated with RQ I to prevent DNA contamination and partially digested with MNase. Protein–RNA complex is puriﬁed by immunoprecipitation by incubating with DynaBeads protein G conjugated with anti-FTO antibody (source: MBL, cat# RN121PW, lot# 1254635), IgG (source: Millipore, cat# 12-370, lot# 3256125 ) or anti-Flag antibody (sigma, cat# F7425, lot# 2323211) at 4 °C for 2 hours. RNA is dephosphorylated at 3’ end and phosphorylated at 5’ end. The protein–RNA complex is separated by 4-12% NuPAGE Bis-Tris gel and the region of the membrane 30 kDa above the protein size is excised. Protein is digested by proteinase K and isolated RNA was used to prepare sequencing library using Balancer sm/miRNA Library Preparation Kit according to the manufacturer’s instructions. The Kit provides one-day single tube procedure that starts with total RNA, goes through adapter ligation, RT, PCR and gel purification, then generates a library ready for Illumina machines. The cDNA libraries were subsequently quantified and sequenced on the Illumina Nextseq platform. Image processing and base-calling were performed by Illumina Pipeline.