Imagequant tl software

Detection of C-circles was performed as previously described^{40 (link)}. Briefly, 30 ng DNA was combined with 10 µl 2X Φ29 Buffer, 7.5 U Φ29 DNA polymerase (NEB), 0.2 mg/ml BSA, 0.1% (v/v) Tween 20, 1 mM each dATP, dGTP and dTTP and incubated at 30 °C for 4 h and 8 h followed by 20 min at 70 °C. Amplification products were deposited on a Hybond N + nylon membrane (Bio-Rad) and developed using the TeloTAGGG Telomere Length Assay Kit (Roche). Chemiluminescent signals was visualized with ChemiDoc XRS system (Bio-Rad) and the intensity of the spots was quantified with ImageQuant TL software (Bio-Rad).

The detection of C-circles was performed as previously described [30 (link)]. Briefly, 30 ng of DNA was combined with 10 μl of 2X Φ29 buffer, 7.5 U of Φ29 DNA polymerase (NEB), 0.2 mg/ml BSA, 0.1% (v/v) Tween 20, and 1 mM each dATP, dGTP, and dTTP and incubated at 30 °C for 4 h or 8 h followed by 20 min at 70 °C. Amplification products were deposited onto a Hybond N+ nylon membrane (Bio-Rad) and developed using the TeloTAGGG Telomere Length Assay Kit (Roche). Chemiluminescent signals were visualized with a ChemiDoc XRS system (Bio-Rad), and the intensity of the spots was quantified with the ImageQuant TL software (Bio-Rad). The 293T and U2OS cell lines were used as negative and positive controls. The intensity of the spots was quantified with ImageJ Fiji (version 1.53c) [31 (link)]. The positive control spot intensity was fixed at 100, and all the other samples were calculated in comparison with the positive control. A sample was considered to be ALT-positive if it had a relative value of > 30, which corresponds to a clearly distinguishable intensity (Additional file 1: Fig. S1c).