Single-cell m⁶A mapping in vivo using picoMeRIP–seq
–1Antibody–bead incubation
Before use, Dynabeads were washed by taking 20 μl of beads and washing them twice with 1× IP buffer by vortexing, quickly centrifuging on a MiniGalaxy and placing on a magnetic rack before discarding the supernatant. In a separate tube, the antibody was diluted by taking 4 µl of anti-mA, 16 µl of 5× IP buffer and 60 µl of nuclease-free water and mixing by gentle vortexing.
For both input and immunoprecipitated RNA samples, nuclease-free water was added to each tube to result in a final volume of 400 μl. Next, 40 μl of 3 M sodium acetate and 10 μl of linear acrylamide 5 mg μl were added, followed by 1,000 μl of ice-cold 100% ethanol. The samples were vigorously vortexed without centrifugation or spinning and immediately placed at –80 °C for at least 2 h or overnight until completely frozen.
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