Fig. 3. Long-read detection of retrotransposition in HeLa cells and MEF-derived bulk miPSC lines.
a Bulk MEFs were reprogrammed by the addition of doxycycline. Oct4-GFP positive miPSCs were then sorted and expanded in serum. Six miPSC lines were reprogrammed and cultured in media containing 100 µM lamivudine (3TC), and six miPSC lines generated without lamivudine (CTRL). DNA was extracted from MEFs and miPSCs and then ONT sequenced. b Top left: retrotransposition indicator plasmid L1-mCherry consists of the pCEP4 backbone (CMV promoter, black; SV40 polyadenylation signal, open lollipop; hygromycin resistance gene HYGR, white) containing a wild-type L1 TF element (5′UTR, light purple; ORFs, dark purple). An mCherry reporter gene equipped with an EF1α promoter and HSVtk polyadenylation signal (black lollipop) is inserted into the L1 3′UTR antisense to the L1. The mCherry sequence is interrupted by an intron in sense orientation relative to the L1, ensuring mCherry expression only upon retrotransposition. bottom left: retrotransposition assay timeline. Cells were split (S), transfected (T), and cultured in hygromycin-containing medium with and without 100 µM 3TC. Retrotransposition efficiency was assessed by flow cytometry 8 days post-transfection (R). top right: fluorescence microscopy images showing representative wells at 8 days post-transfection with L1 TF (left), reverse transcriptase mutant (RT-) L1 TF (middle), and L1 TF treated with 100 µM 3TC (right). Scale bars (white) represent 100 µm. bottom right: Retrotransposition efficiency assessed by flow cytometry, relative to L1 TF. Histogram depicts the mean ± SD of three independent biological replicates (black dots, n = 3) consisting of three technical replicates each. c Target site duplication (TSD) size distributions for L1-mCherry retrotransposition events recovered from HeLa cells via TLDR analysis of ONT sequencing, divided into integrants detected by >1 (top) or 1 (bottom) spanning read. Inset sequence logos120 display the observed integration site motif, as preferred by the L1 endonuclease (EN). d Integrative genomics viewer (IGV)121 inspection of 3 example de novo TE insertions. Cartoons (top) show a full-length (>6 monomers) L1 TF insertion, a heavily 5′ truncated L1 TF insertion, and a SINE B2 insertion, each flanked by TSDs (gray triangles) and a 3′ polyA tract, and integrated at the indicated L1 EN motif. IGV snapshots (bottom) show ONT read alignments from the miPSC line carrying each TE insertion (purple box). e A 5′ truncated intergenic de novo L1 TF insertion. PolyA (An) tract length is indicated immediately 3′ of the L1. PCR validation primers are shown as red arrows. Underneath is shown a PCR validation agarose gel containing the 5′ junction PCR product (red arrow) only in the miPSC line where the L1 was detected by ONT sequencing. Molecular weight (mw) markers are provided at the left of the gel. Note: DNA was obtained from two parental MEF aliquots, one corresponding to miPSC CTRL/3TC lines 1–3 and one to miPSC CTRL/3TC lines 4–6. f TSD size distributions for de novo L1 (top) and SINE (bottom) insertions detected in miPSC lines via ONT sequencing, with inset integration site sequence logo, as per (c). g Putative reprogramming-associated L1-mediated insertion counts detected by ONT sequencing in CTRL and 3TC-treated miPSCs. Replicate (n = 6 per group) data points are marked by black dots and represented as the mean ± SD. Significance was calculated via a two-tailed t test. Source data are provided as a Source Data file.