Typhoon fla 9000 gel imaging scanner

Evaluation of the integration activity of the reconstituted Ty3 intasome was performed through analysis of the DNA products after targeting at the full RNA Polymerase III transcriptional machinery. Samples were collected after recruitment of TFIIIC, TFIIIB and Ty3 intasome to the t(GUC)K target DNA, which was Cy3- or FITC-labelled in the upstream or downstream terminus, respectively (Fig. 1a). Reaction mixtures were prepared in the presence of Cy5-labelled or unlabelled intasomes and, as a control, the assay was also performed in the absence of integration machinery (Fig. 1c, d). In order to analyse the nucleic acid fraction, the protein content was degraded using the broad-spectrum serine protease proteinase K. Thus, the sample was mixed with a proteinase K mixture (2 mg/ml proteinase K, 2% SDS (w/v) and 200 mM EDTA pH 8) in a 10:1.1 volume ratio and incubated for 1.5 h at 37 °C. Then, the cleaved sample was loaded into a 4% agarose gel and identification of the integration products was performed through detection of the FITC, Cy5 and Cy3 fluorophores in a Typhoon FLA 9000 gel imaging scanner (GE Healthcare) (Fig. 1c, d). 50 bp DNA markers (N3236S, New England Biolabs) were used to identify the size of the resulting DNA products (Fig. 1e). All uncropped gel images are available in the Source Data file.

Preparation of chromosomal DNAs and their separation by PFGE were performed as previously described^{26 (link)}. Chromosomal DNAs were prepared in 0.8% low melting agarose gels (Nacalai tesque, 01161-12) and resolved using a CHEF-DRII pulsed-field electrophoresis system (Bio-Rad) under the following conditions. For broad-range PFGE, a 1600 s pulse time at 2 V cm⁻¹ for 42 h followed by a 180 s pulse time at 2.4 V cm⁻¹ for 4 h, at 4 °C in 1× TAE buffer using 0.55% Certified Megabase agarose gel (Bio-Rad, 161-3109)). For short-range PFGE, a 40–70 s pulse time at 4.2 V cm⁻¹ for 24 h, at 4 °C in 0.5× TBE buffer using 0.55% Certified Megabase agarose gel. DNAs were stained with 0.2 µg mL⁻¹ EtBr (Nacalai Tesque, 14631-94) and detected using a Typhoon FLA9000 gel imaging scanner (GE Healthcare). Gel images were processed using ImageJ software or Adobe Photoshop Elements (Adobe, San Jose, CA).