Computational protocol: CpG Demethylation Enhances Alpha-Synuclein Expression and Affects the Pathogenesis of Parkinson's Disease

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Protocol publication

[…] Methprimer (http://www.urogene.org/methprimer/index1.html) was used to design primers for bisulfite sequencing (5′-GGGAGGTTAAGTTAATAGGTGGTAA-3′ and 5′-CCCTCAACTATCTACCCTAAACAAAC-3′).Bisulfite conversion was performed with the EpiTect Bisulfite kit following the manufacturer's “low-concentration” protocol (QIAGEN). For each samples, 100ng of genomic DNA was used and incubated at 95°C for 5min, 60°C for 25min, 95°C for 5min, 60°C for 85min, 95°C for 5min and 60C°C for 175min. Converted product was purified following the instructions of the Epitect Bisulfite kit. One µL from the final 20 µL elute was PCR-amplified with LA Taq (Takara) following the manufacturer's mixture condition and 30 PCR cycles of 98°C 10sec, 55.5°C (for CpG-1) or 63°C (for CpG-2) 30sec, 68°C 60sec. The PCR product was cloned to pCR′-TOPO vector (Invitrogen). At least 20 clones from each experiment were sequenced by M13 reverse or M13 forward (−20) primers. There are 21 CpH sequences in the region. We used sequence results up to 3 unconverted CpHs for further analysis. For each samples, patterns of methylation and unconverted CpHs were used to detect similar clones. Up to four overlapping clones were allowed for measuring CpG methylation. Data analysis and methylation patter figures were generated using QUMA software . […]

Pipeline specifications

Software tools methPrimer, QUMA
Application BS-seq analysis
Diseases Parkinson Disease