Sirna on target plus smartpools

For siRNA knockdown the On-Target Plus (OTP) siRNA Smartpools from Dharmacon (GE) were used, unless otherwise indicated (see Supplementary Table S1). To deplete endogenous RAD51AP1 for rescue experiments and SIM imaging a single siRNA targeting the 3’UTR of RAD51AP1 mRNA (see Supplementary Table S1) synthesized and purchased from Dharmacon (GE). Briefly, 200,000 and 700,000 cells were seeded per well of a 6-well plate and 10cm dish containing growth medium without antibiotics, respectively. ~2hrs later cells were transfected. siRNAs and Dharmafect were diluted in OptiMEM (Life Technologies). A working siRNA concentration of 50nM was used. We used 2.5μL and 5μL Dharmafect transfection reagent per well and 10cm plate, respectively. Transfection medium was replaced with complete culture media 24hrs later or cells were split for desired application and harvested at 72hrs post transfection, unless otherwise indicated.

For siRNA knockdown, the On-Target Plus (OTP) siRNA Smartpools from Dharmacon (Horizon) were used, unless otherwise indicated. To deplete endogenous HIRA for rescue experiments, a single siRNA targeting the 3′ UTR of HIRA messenger RNA was synthesized and purchased from Dharmacon (Horizon). Briefly, 200,000 and 700,000 cells were seeded per well of a 6-well plate and a 10-cm dish containing growth medium without antibiotics, respectively. About 2 h later, cells were transfected. siRNAs and Dharmafect were diluted in OptiMEM (Life Technologies). A working siRNA concentration of 50 nM was used. We used 2.5 μl and 5 μl Dharmafect transfection reagent per 6-well and 10-cm plate, respectively. Transfection medium was replaced with complete culture medium 24 h later, or cells were split for desired application and collected at 72 h post-transfection, unless otherwise indicated. Sequences or Dharmacon catalog numbers of siRNAs used in this study are as follows: HIRA, 5′-GAUGACGACAGUGUUAUCCUU-3′; HIRA 3′ UTR, 5′-GACCUAAGACCUAUGUAAAUU-3′; SAFB1, 5′-UCAAUUUCGUCAGGAUUACUU-3′; CABIN1 no. J-012454-09; UBN1 no. J-014195-05; XRCC1 no. L-009394; RBMX no. L-011691; hnRNPUL1 no. L-004132; FUS no. L-009497; VCP/p97 no. L-008727; ARP2 no. L-012076; ARP3 no. L-012077.