Mirin

Commercially available small molecules used in this study were SCR7 (Tocris; 5342), Azidothymidine (AZT; Tocris), B02 (Sigma; SML0364), 6-Hydroxy-DL-DOPA (DOPA; Tocris), Cyclosporin H (CsH) (Sigma; SML 1575), Mirin (Cayman; 13208), Olaparib (LC Labs; O-9201), AZD7762 (Cayman; 11491), VE822 (Cayman; 24198), RS1 (Calbiochem; 553510), NU7026 (Cayman; 13308), NU7441 (Tocris; 3712), Trichostatin A (TSA) (Cayman; 89730), Pevonedistat (MLN4924) (Selleckchem; S7109), and M3814 (MedKoo; 206478). As a control, this study included the CRISPY Mix (51 (link)), which is the combination of 20 μM NU7026 (NHEJ inhibitor), 0.01 μM TSA (HDAC inhibitor) and 0.5 μM MLN4924 (improves CtIP) (NSC 15520 was not available). Stock solutions were prepared in dimethylsulfoxide (DMSO) (Sigma) and diluted to working concentrations before use. The medium was changed 24 h after the addition of small molecules. We listed the detailed information and working concentrations of small molecules in Supplementary Table S2.

mESCs were treated 40–48 h after passage. mESCs were incubated in culture with 250 µM 5-chloro-2′-deoxyuridine (CldU, Sigma, C6891) for 20 min, washed twice with PBS, incubated with 2 mM hydroxyurea (HU, Sigma, H8627) for 3 h, washed twice with PBS, and incubated with 250 µM 5-Iodo-2′-deoxyuridine (IdU, Sigma I7125) for 20 min. Then, 100 µM IAA (Sigma, I5148), 50 µM mirin (Cayman, 13208), 10 µM PFM01 (Tocris, 622210, Bristol, UK), 10 µM DNA2-IN-C5 (Aobious, AOB9082, Gloucester, MA, USA), 2.5 µM CSN5i-3 (Novartis Pharma, Basel, Switzerland), 25 µM compound 33-11 (ChemBridge Corporation, 6655693, San Diego, CA, USA), or 5 µM ML216 (Aobious, AOB1300) were added at the indicated time points. In place of HU treatment, aphidicolin (Cayman Chemical, 14007) was used at 15 µM. Labeled mESCs were treated with TrypLE and resuspended in PBS at 2 × 10⁵ cells/mL. DNA fiber spreading and immunostaining were performed as previously described [137 (link)]. Primary antibodies used were rat anti-BrdU (CldU) (Abcam, Waltham, USA) and mouse anti-BrdU (IdU) (Becton Dickinson, Franklin Lakes, NJ, USA). Secondary antibodies used were Alexa Fluor anti-rat 568 and Alexa Fluor anti-mouse 488. Antibody information is presented in Supplementary Table S1.