GSM1166135: CTL3; Homo sapiens; OTHER
Medium was removed and cells were lysed directly on the dish with 3 ml Sepasol (Nacalai Tesque, Japan). Total RNA was then extracted following standard protocol with Sepasol. mRNA was further purified using two rounds of Oligotex-dT30 mRNA Purification Kit (Takara Bio, Japan). mRNA was fractionnated using NEBNext Magnesium RNA Fragmentation Module (NEB, Japan) following manufacturer's protocol. All mRNA samples were checked by Bioanalyzer 2100 (Agilent Technologies) to confirm size and quality. In addition to the extraction steps common to all samples, Me-RIP samples were further processed according to the following method. 500 ng fragmented mRNA was incubated in 250 ml of 20% protein A- or G-Agarose slurry in PBS 0.1M, 0.01% Triton X-100, 0.1 mg/ml BSA, 1mM DTT, 0.4 U/ml RNase inhibitor (Toyobo) for 1 h at 4°C for background removal. The RNA/beads slurry was then centrifuged at 700xg, 4°C for 2 min, the RNA supernatant (~200 ml) was used in the next step and the agarose beads pellet discarded. 50% protein A-Agarose slurry in the same buffer was conjugated with anti-m6A rabbit polyclonal antibody (Synaptic Systems, 30 mg antibody/ml slurry) for 3 h at 4°C. Beads were then washed by 5 cycles of re-suspension with >2 volumes of buffer, then centrifugation (700xg, 4°C for 2 min). The final 50% beads-antibody conjugate slurry was then aliquoted into 1.5 ml tubes, 100 ml/tubes; tubes were centrifuged at 700xg, 4°C for 2 min and beads (~50 ml) were re-suspended with the RNA supernatant from the previous step and incubated at 4°C overnight in a tube rotator, with 0.5 mM random decamers (Invitrogen, Japan) in the incubation buffer as blocking reagent. The tubes were then centrifuged at 700xg, 4°C for 2 min. Pellets (methylated RNA) were washed by 5 cycles of re-suspension with at least 10 volumes of buffer and centrifugation at 700xg, 4°C for 2 min. The final pellet (~50 ml) was homogenized with 1 ml Sepasol RNA I super G by vortexing at full speed then kept at -80°C until processed further. RNA was then extracted with a combination of Qiazol/RNeasy micro kit following Qiagen protocol, with Sepasol instead of Qiazol. The RNA eluate (~14 ml) was then used for RNA quantification by Nanodrop and reverse-transcribed using VILO (Invitrogen, Japan). Sequencing libraries were prepared using the NEBNext® Ultra RNA Library Prep Kit with partially modified steps in random primer annealing condition as 65 °C for 15 min.