Total RNAs were isolated from cells and mouse tissues according to manufacturer’s instructions using TRIzol reagent (Cat.# 15596, Life Technologies), then was resuspended in RNA-free water, digested with RQ1 RNase-Free DNase (Cat. # M6101, Promega) for 20 min at 37°C and then subject to phenol/chloroform extraction and ethanol precipitation. The resulted total RNA pallet was resuspended in RNA-free water. PolyA+ RNAs were selected using Dynabeads® mRNA Purification Kit (Cat.# 61006, Invitrogen) according to the manufacturer’s protocol. The PolyA+ RNA was fragmented to 20~80nt nucleotide fragments by alkaline hydrolysis (1mM EDTA, 6mM Na2CO3, 44mM NaHCO3, pH~9.3) at 95°C for 18 minutes, then the RNA fragments precipitated following standard ethanol precipitation. Dynabeads Protein A beads (250ul, Cat. # 10008D, Life Technologies) was pretreated with BSA (Cat. # B9000S, NEB) for 45 minutes at room temperature to reduce background. Anti-m6A rabbit polyclonal antibody (25ug, Cat. # 202003, Synaptic Systems) was then conjugated to Dynabeads Protein at room temperature for 1 hour. The fragmented polyA+ RNAs (3ug) were subject to PAGE selection for 20nt~80nt size and then incubated with the antibody in 1x PXL buffer (1x PBS, 0.1% SDS, 0.5% Sodium Deoxycholate and 0.5% NP-40) supplemented with 2% RNasin® Plus RNase Inhibitor (Cat. # N2615, Promega) for 2 hours at 4°C.  After incubation, the bead and antibody was washed 5 times by 4 different wash buffers: first twice by ice cold Nelson Low Salt Buffer (15mM Tris pH7.5 and 5mM EDTA), once by ice cold Nelson High Salt Buffer (15mM Tris pH7.5, 5mM EDTA, 2.5mM EGTA, 1% Triton X-100, 1% Sodium Deoxycholate, 0.1% SDS and 1M NaCl), once by ice cold Nelson Stringent Wash Buffer (15mM Tris pH7.5, 5mM EDTA, 2.5mM EGTA, 1% Triton X-100, 1% Sodium Deoxycholate, 0.1% SDS, 120mM NaCl and 25mM KCl) and last wash by ice cold NT-2 Buffer (50mM Tris pH7.4, 150mM NaCl, 1mM MgCl2 and 0.05% NP-40). After the stringent wash series, the bead and antibody was split into two halves: one half performed m6A-CLIP leg, and the other half had its bounded RNA eluted with 0.5mg/mL N6-methyladenosine sodium salt (Cat. # M2780, Sigma-Aldrich) at 4°C for 1 hour (m6A-IP leg).  The eluted RNA was ethanol precipitated and re-suspended in RNase-free water for downstream cDNA library preparation. The eluted RNA from m6A-IP step and the input RNA fragments were first treated with T4 PNK to remove its 3’phospho group and then ligated to 68 pmol pre-adenylated DNA linker (L32N from IDT) with T4 RNA ligase2, truncated KQ (Cat. # M0373L, NEB) overnight at 16°C. This ligation mixture was subject to 8% PAGE purification to harvest the ligated product. The purified RNAs from m6A-IP, m6A-CLIP and their input RNA fragments were all subject to the same BrdU-CLIP cDNA library preparation. The detailed BrdU-CLIP cDNA library protocol was described previously (Weyn-Vanhentenryck et al., 2014).
GEO Accession
GSM2300432