Supplementary MaterialsS1 Fig: GFP reporters, integration schematic, and confirmation PCR

Supplementary MaterialsS1 Fig: GFP reporters, integration schematic, and confirmation PCR. products using primers that flank the GFP cassette (RMR1 and RMR2) through the 7 reporter cassette using the indicated oligonucleotide as well as the EJ7ins reporter cassette, expressing the sgRNAs/Cas9 focusing on the 5′ & 3′ sides of the nonhomologous put in. UN, untransfected; +, GFP+ cells enriched by sorting. Primers that amplify Actin had been used like a positive control.(TIF) pgen.1008319.s002.tif (697K) GUID:?27BB6A03-6446-4501-9FE5-21ECF158B689 S3 Fig: Frequencies of RMR events from Fig 3 and Fig 4 complementation analysis, but also like the parental cell line analysis from Fig 2 and Fig 4, and outcomes from mutant cell lines transfected without EV, each normalized to transfection efficiency. (A) Demonstrated Trilostane are frequencies for the parental as well as the cell range for RMR occasions induced using the 5 advantage DSB, and mix of 5 and 3 advantage DSBs. Two independent clones were tested for each reporter in each cell line with four independent replicates for a total = 8, except the parental 23 nt repeat where six independent clones were tested for = 24. Error bars represent SD. * 0.05, ** 0.01, *** 0.005, **** 0.001, parental no EV vs. mutant (No EV), and mutant EV vs. complementation using unpaired 0.05 using unpaired cell line for RMR events induced with the 5 edge DSB, and combination of 5 and 3 edge DSBs. Experiments were performed as in panel (A), except for the 18 nt repeat where four independent clones were tested for = 16. Statistics are as in (A). (C) Shown are frequencies for the parental and the cell line for RMR events induced with the 5 edge DSB, and combination of 5 and 3 edge DSBs. Experiments and statistics were performed as in (A). (D) Shown are frequencies for the parental, cell lines for RMR events induced with the mid-ins DSB. Experiments and statistics were performed as in (A).(TIF) pgen.1008319.s003.tif (1.3M) GUID:?DF3FB1F2-B21C-440A-AFD2-98F1B67B3679 S4 Fig: (A) Frequencies of RMR events from overexpression of POLQ and RAD52 in the parental cell line, normalized to transfection efficiency. The parental reporter cell lines were transfected with an expression vector for the sgRNA(s) and Cas9, as indicated, along with empty vector (EV), POLQ expression vector, or RAD52 expression vector. Error bars represent SD. Two independent clones were tested for MYO9B each reporter with two independent replicates for a total = 4. ? 0.05 (unadjusted 0.05, ** 0.01, EV vs. overexpression (POLQ or RAD52) using unpaired = 4, except parental EV where four replicates were analyzed for = 8. ? 0.05 (unadjusted 0.05, **** 0.001, EV vs. overexpression using unpaired = 4, except DSB 5′ & 3′ edge where four independent replicates were analyzed for = 8. (C) Percentages of GFP+ cells from the non-targeting siRNA (siCTRL) in Fig 5C (left panel) normalized to transfection efficiency including the 12-7-12, 14-7-14, 16-7-16, 18-7-18, and 20-7-20 oligonucleotides. UN, untransfected. Error bars represent SD. Two independent clones were tested with two Trilostane replicates for a total = 4. (D) Percentages of GFP+ cells from Fig 5B complementation analysis, normalized to transfection efficiency, but also including the parental cell line with EV. Error bars represent SD. = 8 for 12-7-12, 14-7-14, 16-7-16, and = 16 for 18-7-18 and 20-7-20. ? 0.05 Trilostane (unadjusted P-value), * 0.05, ** 0.01, *** 0.005, **** 0.001, EV vs. complementation using unpaired = 4. ns, not significant, 14-0-14 vs. no oligonucleotide (none) and LUC-oligo using unpaired 0.05 (unadjusted P-value), * 0.05, ** 0.01, **** 0.001, parental EV vs. mutant EV, and mutant EV vs. complementation using unpaired = 8. * 0.05, ** 0.01, **** 0.001, parental EV vs. mutant EV, and mutant EV vs. complementation using unpaired 0.05 and ** 0.01, parental vs. mutant, and parental siCTRL vs. other siRNA treatments using Fishers exact test. (B) Influence of BRCA2 depletion on replication fork progression without stress and after stress, performed as in Fig 7A and 7B. Parental cells were treated with non-targeting siRNA (siCTRL) or with a pool of four BRCA2 siRNA (siBRCA2), as in Fig 7C. Numbers of fibers analyzed and statistics are as in Fig 7A and 7B.(TIF) pgen.1008319.s006.tif (1.3M) GUID:?BD78A87A-0A73-4655-B038-6E6D03726973 S1 Table: Sequences of sgRNAs and other oligonucleotides. (PDF) pgen.1008319.s007.pdf (141K) GUID:?39D89360-4234-47A2-9617-C8E20CCEFA7E Data Availability StatementAll relevant data are within the manuscript and its Supporting Information data files. Abstract Disrupting either the DNA annealing aspect RAD52 or the A-family DNA polymerase POLQ could cause artificial lethality with flaws in and mutations upstream from the.