E-MTAB-7644
Identification of de novo LINE-1 (L1) retrotransposon integration sites in HeLa S3 cells by ATLAS-seq-neo (unselected insertions)
We used ATLAS-seq-neo to map the sites of integration of an engineered LINE-1 (L1) retrotransposon into the genome of HeLa S3 cells. In brief, we transfected cells with a plasmid-borne L1.3 element carrying a neomycin-resistance-based retrotransposition cassette, as well as a hygromycin-resistance cassette on the plasmid backbone. For this set of experiments, cells were only selected for transfection (hygromycin) but not for retrotransposition (neomycin). Then we prepared ATLAS-seq-neo libraries. Each sample corresponds to an independent transfection and pool of hygromycin-resistant cells. ATLAS-seq-neo relies on the random mechanical fragmentation of the genomic DNA to ensure high-coverage, ligation of adapter sequences, suppression PCR-amplification of the 3' end L1 junction with its flanking genomic sequence, and Ion Torrent sequencing using single-end 400 bp read chemistry. The primer used for suppression PCR specifically targets the engineered element and not endogenous copies as in the original ATLAS-seq protocol (Philippe et al. eLife 2016). For some libraries, the linker-ligated genomic DNA was digested with BamHI, which cuts downstream of L1 polyA site in the plasmid backbone, to limit amplification from the plasmid and enrich for retrotransposition-mediated insertion events into the genomic DNA.
We used ATLAS-seq-neo to map the sites of integration of an engineered LINE-1 (L1) retrotransposon into the genome of HeLa S3 cells. In brief, we transfected cells with a plasmid-borne L1.3 element carrying a neomycin-resistance-based retrotransposition cassette, as well as a hygromycin-resistance cassette on the plasmid backbone. For this set of experiments, cells were only selected for transfection (hygromycin) but not for retrotransposition (neomycin). Then we prepared ATLAS-seq-neo libraries. Each sample corresponds to an independent transfection and pool of hygromycin-resistant cells. ATLAS-seq-neo relies on the random mechanical fragmentation of the genomic DNA to ensure high-coverage, ligation of adapter sequences, suppression PCR-amplification of the 3' end L1 junction with its flanking genomic sequence, and Ion Torrent sequencing using single-end 400 bp read chemistry. The primer used for suppression PCR specifically targets the engineered element and not endogenous copies as in the original ATLAS-seq protocol (Philippe et al. eLife 2016). For some libraries, the linker-ligated genomic DNA was digested with BamHI, which cuts downstream of L1 polyA site in the plasmid backbone, to limit amplification from the plasmid and enrich for retrotransposition-mediated insertion events into the genomic DNA.