GSM5315997: PKKO_CJ_G1; Mus musculus; OTHER
Samples were collected at specified time points for harvested for genomic DNA To study RAG-generated breaks, 20 mg of genomic DNA from v-abl transformed B cells with stable integration of the pMX-INV substrate was used as input. Sonicated DNA was linearly amplified with biotinylated primer (/5BiosA/CAACCACTACCTGAGCACC) and nested amplified with (5' CAAAGACCCCAACGAGAAGC3') primers. Germline sequence was removed with BstBI (NEB) digestion. To study endonuclease-generated breaks, v-abl-transformed B cells were transiently infected with a retrovirus encoding I-PpoI-ER fusion followed by IRES human CD8, 20 hours after infection, half of the cells were arrested in G1 via STI571 (3 µM; Novartis Pharmaceuticals) for 24 hours, and treated with 500nM 4OHT to induce I-PpoI nuclear translocation for 24 hours. The other half was arrested in the S/G2 phase with CDK1 inhibitor (10 mM RO3306) for 16 hours, and 500nM 4OHT to induce I-PpoI cutting for 24 hours. Cells were collected at the end from both conditions for genomic DNA. Here, sonicated DNA was linearly amplified with biotinylated primer (/5BiosG/ACTTAGAACTGGCGCTGAC) and nested amplified with (5' AACCCCAAAGCATCGCGAAG3') primers. Germline sequence was removed with I-PpoI (Promega) digestion. To specifically study the DNA repair junctions on the pMX-INV substrate we added the substrate sequence as an additional chromosome on top of the mm10 genome during the alignment process. high-throughput genome translocation sequencing (HTGTS). Sequenced with MiSeq 500v