FTO CLIP-seq of AHCY WT and Vector in A549 cell line

Collected A549 cells 48 hours post-transfection. CLIP was performed as previously described. Added 8-10 mL PBS to the dish and irradiated the cells at 400 mJ/cm2 and then again at 200 mJ/cm2 in the UV cross-linker on ice. After UV-crosslinking, the cells were collected into a 50mL conical tube and centrifuged at 200g for 5 minutes at 4. Washed the cell pellet in 1ml cold PBS and centrifuged at 1,000g for 5 minutes at 4. Lysed the cell pellet in 1ml 1xPXL (1xPBS [cell culture grade], 1% NP40, 0.5% sodium deoxycholate, and 0.1% SDS) and incubated on ice for 10 minutes. RNase (NEB, M0660S) was used to fragment the RNA. The lysates were then centrifuged in prechilled ultracentrifuge (using 11x34 mm polycarbonate tubes in a TLA120.2 rotor) at 32,000g for 25 minutes at 4. Meanwhile, HA-tagged magnetic beads (MCE, HY-K0201) were washed three times with BWB buffer (1xPBS cell culture grade and 0.02% Tween-20), followed by three washes with 1xPXL. The supernatant of the ultracentrifuged lysates was incubated with the washed beads on a rotator at 4 for 2 hours. The beads were then washed twice with 1xPXL, followed by one wash each with high-salt (1xPBS, 1M NaCl final concentration, including the ~140mM NaCl present in PBS, 1% NP40, 0.5% sodium deoxycholate, and 0.1% SDS), high-stringency (15mM Tris-HCl pH 7.5, 5mM EDTA pH 8.0, 2.5mM EGTA pH 8.0, 1% NP40, 1% sodium deoxycholate, 0.1% SDS, 120mM NaCl, and 25mM KCl), and low-salt (1 mM Tris-HCl pH 7.5 and 5mM EDTA) wash buffers. The beads were then washed twice with 1xPNK (50mM Tris-HCl pH 7.5, 10 mM MgCl2, and 0.5% NP40) buffer. Then the samples were sent for sequencing.