Single-cell m6A profiling reveals mRNA methylation heterogeneity and unique cellular m6A signatures

We used DART-seq to map m6A methylation of RNA in single HEK293T cells. We also used DART-seq to map m6A from bulk RNA from HEK293T cells. Using the 10X Genomics and SMART-seq2 platforms, we sequenced a total of 19,533 experimental and control cells using the 10X Genomics platform, and 1,471 experimental and control cells using SMART-seq2. We then used a Bullseye, a computational pipeline developed within the lab, to identify m6A sites from the C-to-U mutations in bulk and single-cell datasets. We find that most m6A methylation is highly heterogenous from cell-to-cell. RNAs containing m6A methylation, are infrequently methylated, and that most individual sites are rarely methylated within the population. Additionally, we are able to identify differentially methylated RNAs in different cellular states from within a single population, and use m6A methylation information to perform clustering of single cells to find a source of novel cellular heterogeneity. Bulk DART-seq: We performed bulk RNA-seq on RNA from HEK293T cells expressing APOBEC1-YTH. We also sequenced RNA from cells expressing APOBEC1-YTHmut, which serves as controls. There are 3 biological replicates included for each of these samples. 10x Genomics single-cell DART-seq: We performed 10x Genomics 3' end RNA-seq (3 biological replicates each, targeting ~80,000 reads per cell) on HEK293T cells expressing APOBEC1-YTH or APOBEC1-YTHmut (control cells). A total of 10,352 APOBEC1-YTH cells and 9,151 APOBEC1-YTHmut cells passed QC filters. SMART-seq2 single-cell DART-seq: We generated SMART-seq libraries from single APOBEC1-YTH-expressing HEK293T cells and APOBEC1-YTHmut-expressing HEK293T cells (as controls). Cells were sequenced to a depth of about 5,000,000 reads per library using 150bp paired-end sequencing.