φ29 buffer

CC assay was performed as described previously. Genomic DNA was purified, digested with AluI and MboI and cleaned up by phenol-chloroform extraction and precipitation. DNA was diluted in ultraclean water and concentrations were exhaustively measured to the indicated quantity (30, 15, 7.5ng) using a Nanodrop (ThermoFisher). Samples (10 μ.l) were combined with 10 μL BSA (NEB; 0.2 mg/ml), 0.1% Tween, 0.2mM each dATP, dGTP, dTTP and 1 × Φ29 Buffer (NEB) in the presence or absence of 7.5U ΦDNA polymerase (NEB). Samples were incubated at 30°C for 8hrs and then at 65°C for 20mins. Reaction products were diluted to 100 μL with 2 × SSC and dot-blotted onto a 2 × SSC-soaked nylon membrane. DNA was UV cross-linked onto the membrane and hybridized with a P³² end-labeled TelC (CCCTAA)₄ oligo probe to detect C-circle amplification products. All blots were washed, exposed to PhosphoImager screens, scanned using a Typhoon 9400 PhosphoImager (GE Healthcare) and quantified with ImageJ. In all reactions Φ29 was omitted as a negative control DNA was used.

C-circle assay was performed as previously described (Lau et al., 2013 (link)). Genomic DNA was extracted with PureLink Genomic DNA Mini Kit (K182002). Diluted DNA (16 ng) was combined with 0.2 mg/ml BSA, 0.1% Tween, 4mM dithiothreitol (DTT), 1 mM each dNTP without dCTP, 1× φ29 Buffer (NEB) and 7.5 ∪ φ29 DNA polymerase (NEB). Samples were incubated for 8 h at 30°C followed by 20 min at 65°C. For RCA(+) samples, the assay was done with 429 DNA polymerase. For RCA(−) samples, the assay was done without φ29 DNA polymerase. The levels of telomeric DNA in RCA(−) and RCA(+) samples were quantified by qPCR. C-circles are calculated by ΔCt (RCA+)/ΔCt (RCA−). For each control or knockdown condition, triplicated samples were used for the quantification of C-circles. The primers used in this study are shown in Table S2.

C-circle assay is a specific, quantitative, and responsive indicator of alternative lengthening of telomere (ALT) activity levels, the protocol for C-circle amplification employed was slightly modified from that performed in Henson and colleagues (24 (link)). Briefly, 400-ng genomic DNA was digested with Hinf I and Rsa I restriction enzymes at 37°C overnight and purified by phenol-chloroform extraction. Genomic DNA from ALT-positive (U2OS) cells was used as a positive control. DNA was diluted in double distilled water. Samples (10 µL) were combined with 10 µL 1× Φ29 buffer (New England Biolabs, Beverly, MA), containing bovine serum albumin, 0.2 mM each d ATP, d GTP, d TTP, and incubated in the presence or absence of 5U ΦDNA polymerase (New England Biolabs, Beverly, MA) at 30°C for 12 hours and then at 65°C for 20 minutes. Added 60-ng reaction products to 200 µL 6× SSC and dot-blotted onto a 6× SSC-soaked nylon membrane. DNA was cross-linked onto the membrane and hybridized with a DIG-labeled probe (CCCTAA)₄ to detect C-circle amplification products. Blots were washed, exposed to Tanon 5200 (Tanon Science and Technology Co., Ltd, Shanghai, China), and quantified using ImageJ.

Cells were treated with 5-, 10- or 20 min dye and light. 24 h post treatment cells were harvested, and genomic DNA was purified, digested with AluI and MboI and cleaned up by phenol-chloroform extraction and precipitation. DNA concentrations were exhaustively measured to the indicated quantity (30ng) using a Nanodrop (Thermo Fisher). Samples (10 μL) were combined with 10 μL BSA (NEB; 0.2 mg/mL), 0.1% Tween, 0.2 mM each of dATP, dGTP, dTTP and 1× Φ29 Buffer (NEB) in the presence or absence of 7.5 U ΦDNA polymerase (NEB). Samples were incubated at 30°C for 8 h and then at 65°C for 20 min. Reaction products were diluted to 100 μL with 2× SSC and dot-blotted onto a 2× SSC-soaked nylon membrane. DNA was ultraviolet (UV) crosslinked onto the membrane and hybridized with a P^{32 (link)} end-labeled (CCCTAA)₄ oligonucleotide probe to detect C-circle amplification products. All blots were washed, exposed to PhosphoImager screens, scanned using a Typhoon 9400 PhosphoImager (GE Healthcare) and quantified with ImageJ. In all reactions, when Φ29 was omitted as a negative control DNA was used.