In vitro DART-Sanger Sequencing of APOBEC-YTH

APOBEC-YTH and APOBEC-YTH^mut were purified and in vitro DART-Sanger sequencing assays were performed as previously described (Tegowski et al., 2022 (link)) with minor modifications. Briefly, total RNA was isolated from adult heads with TRIzol (Invitrogen) and treated with DNase I (NEB). RNA was isolated once more with TRIzol (Invitrogen) to remove DNase I and DNase I Buffer (NEB). Next, 200 ng of purified RNA from Drosophila heads was incubated with 1000 ng of purified DART protein in DART buffer (10 mM Tris-HCl, pH 7.4, 50 mM KCl, 0.1 M ZnCl₂) and 1 µL of RNaseOUT (Invitrogen) in a total volume of 200 µL for 4 hr at 37°C. RNA was isolated with the QIAGEN Plus Micro Kit (QIAGEN) and stored at –80°C before being thawed for downstream Sanger sequencing analysis. cDNA was made using iScript Reverse Transcription Supermix (Bio-Rad). PCR amplification of Sxl pre-mRNA was carried out with Phusion High Fidelity PCR Kit (NEB). The resulting PCR product was PCR-purified using the QIAGEN PCR Purification Kit (QIAGEN). Samples were submitted for Sanger sequencing (McLabs) and %C-to-U editing was quantified using EditR software (Kluesner et al., 2018 (link)).