Fluorchem q imager

Proteins from tissues or cells were extracted in RIPA buffer (Sigma-Aldrich, R0278) freshly supplemented with protease inhibitor cocktails (Roche, 11836170001) and phosphatase inhibitor cocktails (Roche, PhosSTOP, 04906837001). Protein concentrations were quantified using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, 23227). Equal amounts of total protein lysates were analyzed by standard Western blot procedures using either the Mini-Protean tris-glycine extended gels or the Criterion tris-glycine extended Midi Protein gels (Bio-Rad). Protein bands were developed using the ECL substrate (Millipore, WBLUC0500) and visualized by FluorChem Q Imager (Alpha Innotech) followed by densitometry quantification using the ImageJ software (80 (link)). Antibody information is provided in Table S6.

T5 Exonuclease assay: 8 nM 3′-Cy3 ssDNA (RP540Cy3) in buffer (50 mM Potassium Acetate, 20 mM Tris-acetate, pH 7.9,10 mM Magnesium Acetate, 1 mM DTT, 30 mM NaCl) was mixed with 10 nM PolθΔcen or Polθ-pol and was preincubated for 10 min at 37 °C. After initial incubation, T5 Exonuclease (New England Biolabs) to a final concentration of 0.1 U/ul or 0.5 U/ul was added to the reactions as indicated and incubated for 15 min at 37 °C. Reactions were terminated by the addition of denaturing stop buffer (90% formamide and 50 mM EDTA). DNA was resolved in denaturing 15% polyacrylamide gels and imaged at the Cy3 wavelength using FluorChem Q imager (Alpha Innotech). EcoRI endonuclease assay: 5 nM 5′-³²P radiolabeled pssDNA (RP538/RP539) in buffer (25 mM Tris-HCl, pH 7.5, 1 mM DTT, 0.01% NP-40, 0.1 mg/ml BSA, 10% glycerol, 10 mM MgCl₂, 2 mM ATP, 30 mM NaCl) was incubated with or without indicated Polθ enzyme for 15 min at 37 °C. After initial incubation, 4 units of EcoRI were added as indicated and incubated for 10 min at 37 °C. Reactions were terminated by the addition of non-denaturing stop buffer (100 mM Tris-HCl, pH 7.5, 10 mg/ml proteinase K, 80 mM EDTA, and 0.5% SDS) and incubated at 37 °C for at least 15–20 min. DNA was resolved in non-denaturing 15% polyacrylamide gels and analyzed by phosphorimager (Fujifilm FLA 7000).

Transgenic plant lines were generated using the Agrobacterium floral dip method with the Col0 rdr6-11 silencing mutant (Peragine et al., 2004 (link)) as described previously (Deuschle et al., 2006 (link)). Transformants were selected on agar plates containing ½ × MS medium with BASTA. Fluorescence expression of transformants on agar plates was analyzed using a FluorChem Q imager (Alpha Innotech, San Leandro, CA) with CY2 excitation and emission and the following settings: 12 s exposure time, normal speed, ultra resolution and level 2 noise reduction. All ABACUS plant lines are summarized in Supplementary file 1.