C15200081

PA-m⁶A-seq and PA-m⁵C-seq were performed as previously described (8 – (link)10 (link)). Briefly, 3T3 cells were infected with MLV as described above. At 48 hpi, cells were pulsed with 100 mM 4-thiouridine (4SU). After a further 24 h, total cellular RNA was extracted from the MLV-infected 3T3 cells using TRIzol, while MLV gRNA was extracted from virions that were collected by ultracentrifugation of the supernatant media through a 20% sucrose cushion. Total cellular poly(A)⁺ RNA was purified using oligo(dT) magnetic beads (AM1922; Invitrogen) and 10 μg of poly(A)⁺ RNA or virion gRNA was then used according to the previously reported PA-m⁶A-seq protocol (10 (link), 25 (link)) using either an m⁶A-specific (202111; Synaptic Systems) or m⁵C-specific (C15200081; Diagenode) polyclonal antibody.

Serially diluted genomic DNA was denatured and loaded onto nitrocellulose membrane using a slot blot apparatus (Biorad). After washing with 4X saline sodium citrate, the membrane was subjected to UV crosslinking and probed with a 5-methylcytosine antibody (C15200081, Diagenode). After chemiluminescence detection, the membrane was stained with 0.2% methylene blue in 0.3M sodium acetate (pH 5.2) as a loading control for the slot blot.