Table 7 Validation of insertions through spanning 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 25uL PCR reactions with ExTaq HS following manufacturer’s instructions.

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 10-minute extension for 30 cycles.

Critical: It is necessary to use an extension time long enough to amplify a full length, 6kb L1 insertion.

5. Run the PCR product on a 1% agarose gel and excise the band containing the filled allele (see Fig. 3a).

Troubleshooting: If no filled band occurs, we recommend trying a 3’ L1 specific PCR. (see Table 8).

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

7. Sanger sequence the purified DNA using both the forward and reverse PCR primer