Separation of bona fide and passenger transcripts TE elements can be detected via template-switch oligo (TSO)–assisted cDNA synthesis. (A, left) Diagram of short reads generated by sequencing bona fide TE elements (green). (Right) Filtering for reads that are linked to the TSO will identify reads that capture the 5′-end of the TE. (B, left) Sequencing cDNA from a Pol II–derived gene with a passenger TE element in the intron (red). (Right) The TSO is bound to the 5′-end of the gene, not the TE. Note that both bona fide and passenger elements generate identical short read information and cannot be distinguished without additional information about the promoter sequence at the 5′-end. (C) BonaFide-TEseq workflow begins with (1) first-strand cDNA synthesis with a TSO, ensuring long first-strand synthesis times to extend to the end of long transcripts. This synthesis step is compatible with both single-cell and bulk RNA-sequencing (RNA-seq) libraries. (2) Sequencing the libraries, in this case with Illumina short reads. (3) All reads are split into separate files based on the capture of the TSO sequence at the 5′-end. (4) Each file: +TSO reads, −TSO reads, and all reads are trimmed of low-quality bases and of the TSO adapter sequence. (5a) Aligning the +TSO and −TSO read files against the Repbase reference for the corresponding lineage, in this case, rodent. (5b) Aligning all trimmed reads to the transcriptome reference using standard procedures such as RSEM. The outputs at the end of this procedure are three separate counts files: +TSO (bona fide TEs), −TSO (passenger TEs), and a standard gene expression matrix. (D) Proportion of nuclei with detectable expression classified as passenger (left) or bona fide (right) TEs within each annotated family. (LTR) Long terminal repeat; (RC) rolling circle; (Sat.) satellite; (scRNA) small cytoplasmic RNA.
