BLV Provirus Integration Site Amplification

The integration sites of BLV provirus were amplified by RAISING as previously described (14 (link)), with some modifications. The primers and reagents used in each step are shown in Tables S1 and 2 in the supplemental material. The reaction conditions of each step are shown in Table S3. Briefly, single-stranded DNA (ssDNA) of the 3′ LTR region of the BLV provirus and the downstream region of the host genome was synthesized from the extracted genomic DNA using the primer BLV-F1 and KOD-Plus-Neo DNA polymerase (Toyobo, Osaka, Japan). The synthesized ssDNA was purified using a Monarch PCR & DNA Cleanup Kit (New England Biolabs, Ipswich, MA, USA) and was eluted in ultrapure water. Then, poly(A) and poly(G) tails were added at the 3′ end of the purified ssDNA by terminal transferase (New England Biolabs). The double-stranded DNA was then synthesized and amplified by PCR from the poly(AG)-tailed ssDNA using the primers BLV-F2 and NV-oligo-dT-ADP1 and Q5 Hot Start High-Fidelity DNA polymerase (New England Biolabs). The second PCR was performed using diluted PCR products, the primers BLV-F3 and ADP1-HTS-R1, and KOD-Plus-Neo DNA polymerase (Toyobo).