Table 8 Validation of insertions and identification of 3’ transduction events through L1-specific 3’ PCR and Sanger sequencing (Timing: variable)

1. Design flanking primers around L1 insertion site.

Critical: Each primer should be at least 100bp away from insertion site. Avoid placing primers in repetitive DNA.

2. Set up duplicate 25uL PCR reactions with ExTaq HS following manufacturer’s instructions. Each reaction should contain one flanking primer paired with the L1 specific primer from vectorette PCR.

Critical: It is important to use a robust polymerase to extend through the L1 poly-A tail.

3. Use 50ng of gDNA as template.

Critical: Use the same high quality gDNA that served as starting material for TIPseq

4. Run the PCR in a thermal cycler with heated lid using a 60°C annealing temperature and at least a 30-second extension for 30 cycles.

Critical: It is important to use a slightly higher annealing temperature and shorter extension time to reduce the amount of off-target L1 binding and amplification.

5. Run the PCR product on a 1% agarose gel and excise the band from the successful reaction.

Critical: Only one of the two PCR reactions should produce a band. A plus stranded L1 insertion will produce a band in the reverse primer reaction, and a minus stranded L1 will produce a band in the forward primer reaction. The size of the band should equal the distance from the genomic primer to the L1 insertion site plus 150bp of L1 and polyA sequence. A band larger than expected could indicate a 3’ transduction event has occurred (See Fig. 3b)

6. Purify the excised DNA using Zymoclean Gel DNA Recovery Kit following the manufacturer’s instructions.

7. Sanger sequence the purified DNA using the L1 primer and either the forward or the reverse genomic primer, depending on which reaction was successful.

Critical: It may be necessary to use internal primers to sequence through the product completely.