GSM8197732: B6AIDKOWT45A; Mus musculus; OTHER
For MiSeq sequencing, genomic DNA extracted from tumors induced by TBLV-WT and TBLV-SD at the higher dose (3 tumors each) in wild-type and Aicda-/- B6 mice was used for PCR with primers env7254(+) (5'-ATC GCC TTT AAG AAG GAC GCC TTC T-3') and LTR9604(-) (5'-GGA AAC CAC TTG TCT CAC ATC-3'), resulting in ~2 kb amplicon of the envelope-LTR junction region. Reactions were performed with JumpStart RED Accutaq LA polymerase (Sigma-Aldrich) in the supplied buffer, 1 µg of tumor DNA, 50 pmol of each primer, and 0.5 mM deoxynucleoside triphosphates in 20 µl.  PCR parameters were: 94oC for 1 min, then 10 cycles at 94oC for 10 sec, 53oC for 30 sec, and 68oC for 2 min followed by 25 cycles of 95oC for 15 sec, 50oC for 30 sec, and 68oC for 2 min with a final incubation at 68oC for 7 min. Sixteen independent reactions for each tumor sample were pooled, and DNA was sheared to a size of approximately 400 bp using a Covaris ultrasonicator at the UT Austin Genomic Sequencing and Analysis Facility (GSAF). For Nanopore sequencing, genomic DNA extracted from tumors from B6, B6 AID (Aicda)-knockout, or B6 mA3-AID double-knockout mice induced by TBLV-WT or TBLV-SD was used for PCR with primers env7254(+) (5'-ATC GCC TTT AAG AAG GAC GCC TTC T-3') and LTR9604(-) (5'-GGA AAC CAC TTG TCT CAC ATC-3') for the region spanning the envelope-LTR junction. PCRs were performed in 25 µl with JumpStart RED Accutaq LA polymerase (Sigma-Aldrich, cat# D8045) in the supplied buffer, 500 ng of tumor DNA, 25 to 50 pmol of each primer, and 0.5 mM deoxynucleoside triphosphates.  PCR parameters were: 94oC for 1 min, 10 cycles at 94oC for 10 sec, 53oC for 30 sec, 68oC for 2 min followed by 25 cycles of 95oC for 15 sec, 50oC for 30 sec, and 68oC for 2 min with a final incubation at 68oC for 7 min. The fragment size for the env-LTR junction was ~2kb. For MiSeq sequencing, fragmented DNA was end repaired and used for dA-tailing and ligation with Illumina adapters (unique to each tumor sample). Adapter-ligated DNA was amplified using these PCR parameters: 98oC for 30 sec, 6 cycles at 98oC for 10 sec, 65oC for 30 sec, and 72oC for 30 sec with a final incubation at 72oC for 5 min. Library preparations were submitted for sequencing on the MiSeq platform at the UT Austin Genome Sequencing and Analysis Facility (GSAF). Nanopore sequencing was employed for high-throughput sequence analysis of TBLV proviruses from the tumors induced by low-dose infection. The extracted PCR fragment used for Sanger sequencing was purified using a Zymoclean Gel DNA Recovery Kit (Zymo Research, cat# D4002). DNA (~1 µg) was prepared for Nanopore long amplicon sequencing by the University of Wisconsin Biotechnology Center.