Typhoon phosphorimager scanner system

1×10⁶ cells were collected and washed with PBS. DNA was isolated using the manufacturer’s instructions (Qiagen, Valencia, CA). 2.5µg DNA was digested with six different restriction enzymes (HhaI, HinfI, MspI, HaeIII, RsaI, AluI) (New England Bio, Ipswich, MA) and incubated at 37°C overnight. Digested DNA was separated on a 0.7% agarose gel overnight at 70 V. The terminal restriction fragment (TRF) gel was placed in denaturating solution for 20 minutes (0.5M NaOH, 1.5M NaCl, pH 13.2) and dried on Whatman 3MM paper under vacuum for 3 hours at 56°C. The gel was then placed for 15 minutes in neutralization buffer (1.5M NaCl, 0.5M Tris-HCl, pH 8.0) and then probed with a radiolabeled telomeric probe (C-rich) for 16 hours at 42°C in 5× SSC buffer, 5×Denhardt’s solution, 10mMl/L Na₂HPO₄, and 1mM/L Na₂H₂P₂0₇. The gel was washed once with 2× SSC, 0.1% SDS, twice with 0.5× SSC, 0.1% SDS and then twice with 0.5× SSC, 1% SDS at room temperature for 15 minutes. Gels were exposed to a PhosphorImager screen overnight and analyzed using a Typhoon PhosphorImager scanner system (Molecular Dynamics). ImageQuant and graphpad prism were used to determine average telomere length of cells.

Telomerase activity was measured by the TRAP assay (Telomeric Repeat Amplification Protocol) (35 (link)). Briefly, HCT116 cells were treated with 1 or 10µM 6-thio-dG for 1 week. 1×10⁵ cells were then collected and lysed with ice-cold NP-40 lysis buffer (10mM Tris–HCl pH 8.0, 1.0mM MgCl₂, 1mM EDTA, 1% NP-40, 0.25mM sodium deoxycholate, 10% glycerol, 150mM NaCl, 5mM β-mercaptoethanol) for 30 minutes. One microliter cellular lysate was used for each reaction. Hela cells were used as a positive control and lysis buffer was used as a negative control. Samples were prepared and then the telomerase extension products were amplified using PCR (95°C for 5 minutes to inactivate telomerase, then 95°C for 30 seconds, 52°C for 30 seconds, 72°C for 30 seconds; 24 cycles). Samples were run on a 10% non-denaturating acrylamide gel and visualized using a Typhoon PhosphorImager scanner system (Molecular Dynamics, GE Healthcare, Piscataway, NJ) that is capable of reading Cy5 fluorescence.