Isoform characterization of m6A in single cells identifies its role in RNA surveillance [m6A-isoSC-seq(nanopore)]
The distribution of m6A across various RNA isoforms and its heterogeneity within single cells are still not well understood. Here, we develop m6A-isoSC-seq, which employs both Oxford Nanopore long-read and Illumina short-read sequencing on the same 10x Genomics single-cell cDNA library with APOBEC1-YTH induced C-to-U mutations near m6A sites. Through m6A-isoSC-seq on a pooled sample of three cell line origins, we unveil a profound degree of m6A heterogeneity at both the isoform and single-cell levels. Through comparisons across single cells, we identify widespread specific m6A methylation on certain RNA isoforms, usually those mis-processed RNA isoforms. Compared to the coding isoforms of the same genes, the expression of highly methylated misprocessed RNA isoforms is more sensitive to METTL3 depletion. These mis-processed RNAs tend to have excessive m6A sites in coding regions, which are targets of CDS-m6A decay (CMD). This study offers undocumented insights into the role of m6A on RNA surveillance. Overall design: The pcDNA3.1-YTH-APOBEC-T2A-EGFP construct was transfected into HEK293T cells, HepG2 cells, and HeLa cells, respectively, using Lipofiter 3.0 (Hanbio) according to the manufacturer's instructions. The medium was refreshed 6 hours after transfection, and the EGFP-positive cells of the three cell lines were isolated, respectively, through cytometry 3 day after transfection. The EGFP-positive HEK293T, HepG2, and HeLa cells were mixed together in a 1700:1700:1700 ratio and then isolated to create cell suspension. The mixed single-cell suspension was utilized to generate a barcoded single-cell RNA-seq library using the 10x Genomics 3' gene expression kit (v3) following the recommended protocol provided by the manufacturer. The other half of the full-length cDNA generated from 10x Genomics was amplified by five cycles of PCR with TruSeq read 1 forward primer 5'-NNN CTA CAC GAC GCT CTT CCG ATC T-3' and 3' partial TSO reverse primer 5'-NNN AAG CAG TGG TAT CAA CGC AGA GTA CAT-3' for Nanopore sequencing. After that, amplified full-length cDNA was treated with 0.55x SPRIselect to remove PCR primers and the cDNAs with lengths shorter than 1000bp. The purified cDNA reagent into a final elution of 51?µL in Buffer EB to allow for adequate template/volume for the necessary assessment of quality control (QC) metrics and PromethION library preparation. Single cell nanopore sequencing library was prepared with the Oxford Nanopore LSK-110 kit (PCR free) and sequenced on PromethION following the manufacturer's instructions to generated 339 million long reads derived from about 5000 cells.