TeSLA measurements were performed as described in Lai et al. (2017)84 (link). Briefly, 50 ng of genomic DNA were mixed with 2 mM ATP, 0.5 μl T4 ligase (NEB), and Telo1-6 ligation oligos at 10 nM each in CutSmart buffer 1X (NEB), and incubated overnight at 35 °C. The mixture was digested with CviAII, MseI, NdeI and BfaI (NEB) to generate DNA fragments with 5′ AT and TA overhangs. The 5’ phosphate of these DNA fragments was removed using the shrimp alkaline phosphatase (rSAP, NEB). The DNA mixture was then incubated overnight at 16 °C with T4 DNA ligase, 1 mM ATP, 1 μM of AT adapter, and 1 μM of TA adapter in 1× CutSmart buffer.
Multiple PCRs were performed using FailSafe Enzyme Mix (Lucigen) with 1× FailSafe buffer H containing 0.25 μM AP/TeSLA-TP primers and 40 pg of ligated DNA. PCR products were loaded on a 0.85% agarose gel and run for 19 h at 1.5 V/cm. After gel electrophoresis, the amplified telomeres were detected by Southern blot.
Multiple PCRs were performed using FailSafe Enzyme Mix (Lucigen) with 1× FailSafe buffer H containing 0.25 μM AP/TeSLA-TP primers and 40 pg of ligated DNA. PCR products were loaded on a 0.85% agarose gel and run for 19 h at 1.5 V/cm. After gel electrophoresis, the amplified telomeres were detected by Southern blot.
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