Sequencing data of DNA repair substrate plasmid by TMEJ

TMEJ assay in nuclear extracts.
The assay was performed as described by Dutta et al. (Dutta et al., 2017) with modifications. Briefly, exponentially growing U2OS cells transiently expressing POLQ-Flag-HA in 60 mm plates (90% confluent) were irradiated with X-rays (10Gy). After indicated time points of incubation, the irradiated and control cells were harvested for preparation of nuclear extracts. 100ng I-SceI digested pBabehygro-EGFP-MMEJ (Truong et al., 2013), the repair substrate, mixed with 100μl nuclear extracts 30 min with gentle shaking at 30oC followed by incubation at 15 h at 16oC. 10μl of mixture were used to XL10- gold ultracompetent E. coli (Agilent Technologies) following the manufacturer’s protocol. The colonies in each agar plate were counted and submitted for PCR + sequence analysis of the product using the PCR primers: 5′-ACGGGGTCATTAGTTCATAGCCCA-3′, and 5′-GGGATTTTGCCGATTTCGGCC-3′; and the sequencing primer: 5'-ATGGTGAGCAAGGGCGAGGAG-3' (Genewiz Inc.).