Ez dna methlyation lightning kit

Genomic DNA was extracted from decitabine or DMSO -treated cells using the EZNA Tissue DNA Kit (#D3396-02, Omega Bio-tek) and subjected to sodium bisulfite modification using EZ DNA Methlyation Lightning Kit (#D5031, Zymo Research) according to the manufacturer’s instructions. The TERT promoter region was amplified by PCR using ZymoTaq™ PreMix (#E2004, Zymo Research) with two pairs of primers. The primer pair 5’- GGAGGAGGYGGAGTTGGAAGGTGAAGGGGTAGGA-3’ and 5’- CCTCCACATCATAACCCCTCCCTCRAATTACCCCACA-3’ was used for amplifying the -175 to -466 bp of TERT promoter, the primer pair 5’- TGTGGGGTAAttyGAGGGAGGGGttATGATGTGGAGG-3’ and 5’- CCTaaCTCCATTTCCCACCCTTTCTCrAC-3’ was used for amplifying the -430 to -840 region of TERT promoter. The PCR products were then cloned to T vector and subjected to Sanger sequencing.

For the bisulfite reaction, 1000 ng of genomic DNA was converted using a EZ DNA Methlyation-Lightning Kit (Zymo Research, USA) following the manufacturer’s instructions. Then 2 µL of converted products were amplified using Q5U Hot Start High-Fidelity DNA Polymerase (NEB, USA) according to the manufacturer’s instructions. PAS-specific PCR was performed in a total volume of 50 µL as follows: 30 s at 98 °C, 35 × (10 s at 98 °C, 30 s at 65 °C, 30 s at 72 °C), and 2 min at 72 °C. The 4qA-allele-specific primers were from Calandra et al. [23 (link)]. The obtained PCR products were purified using a FastPure Gel DNA Extraction Mini Kit (Vazyme, China). Purified PCR products were cloned into a pCE2 TA/Blunt-Zero vector using a 5 min TA/Blunt-Zero Cloning Kit (Vazyme, China) and transformed into Escherichia coli DH5α Electro-Cells.
At least 50 clones were chosen at random from each sample, and individual clones were sent for Sanger sequencing (Tsingke Biological Technology, China). Ten previously reported CpG sites were included as methylation markers. The methylation level for each site was calculated as ratio of methylated sites to total sites. The mean methylation level for the 10 CpG sites was calculated as the average level across the 10 sites. A schematic diagram of the 10 CpG sites is shown in Fig. 1B.