GSM8457961: WT replicate 2 IP; Mus musculus; OTHER
To perform MeRIP-seq on BMDMs, start by enriching PolyA RNA from at least 10 µg of total RNA using the DYNABEADS MRNA PURIFICATION Kit (61006) according to the manufacturer's instructions, followed by purification with VAHTS RNA Clean Beads (N412-02) and elution in 10-20 µl of nuclease-free water. Fragment the eluted RNA at 94°C for 30 seconds using Thermo Fisher Scientific RNA Fragmentation Reagents (AM8740) and purify the fragmented RNA using the ZYMO RNA Clean & Concentrator Kit (R1016). Proceed with the m6A RNA immunoprecipitation (IP) according to the established protocol, ensuring optimal antibody binding and RNA recovery. Following IP, construct the RNA library using the NEBNext Ultra II Directional RNA Library Prep Kit (NEB, E7760S) and purify the library with VAHTS DNA Clean Beads (N411-02). Finally, examine the purified library on a 2% agarose gel, aiming for an ideal library size of approximately 250 bp.