Improved m6A bulk RNA-seq detection in HEK293T cells. (A) Schematic diagram of m6A detection with bulk RNA-seq in cultured cells. The YTH protein domain binds to m6A. When bound to APOBEC1, the APOBEC1 protein converts C-to-U in the vicinity of m6A. This results in a C-to-T mutation in cDNA. C-to-T mutations detected by RNA sequencing are indicative of m6A RNA modifications. (B, left) Immunofluorescence (IF) of HEK293T cells transfected with Apobec1-Yth-HA-Egfp (Yth-HA-E) or Egfp-Apobec1-Yth-HA (E-YTH-HA). Scale bar, 20 μm. Representative images are shown. (Right) Quantification of EGFP and HA overlap. (PCC) Pearson correlation coefficient. (C) Number of C-to-T editing events identified in each bulk RNA-seq HEK293T cell replicate for Yth-E and E-Yth plasmids. Editing events identified in at least two replicates were considered for downstream analyses. The data were obtained following Apobec1-Yth-Egfp or Egfp-Apobec1-Yth transfection and EGFP FACS sorting. n = 3. (Rep) separately cultured replicate. (D) Metagene analysis showing m6A site counts along transcripts for Yth-E and E-Yth bulk RNA-seq results. Nine percent of all m6A sites occur in the first 10% of the 3′ UTR following the TTS for YTH-E and 11% for E-YTH, respectively. Shown percentage indicates number of m6A sites upstream of, within, and downstream from coding sequence (CDS). (E) Metagene analysis showing m6A density 500 nt 5′ and 500 nt 3′ from stop codon (0 nt) for YTH-E and E-YTH.
