Computational protocol: ZNF300 tight self-regulation and functioning through DNA methylation and histone acetylation

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Protocol publication

[…] 293T cells were transfected with pGL3-ZNF300pro-luc plasmids with or without zinc finger domain-encoding region of ZNF300 on the downstream. The plasmid DNA was extracted from the transfected cells according to the manufacturer’s instructions and treated with sodium bisulfite using the EZ DNA Methylation-Gold Kit™ following the manufacturer’s guidelines. One to two ng of sodium bisulfite-converted plasmid DNA was used as a template for methylation specific PCR (MSP). For MSP we designed primer pairs for ZNF300 gene promoter. Primers for DNA analysis were designed using the Methprimer Software (http://www.urogene.org/methprimer/). PCR was conducted using a pfu DNA polymerase (Thermo Scientific), with an initial denaturation step at 95 °C for 10 min, followed by 35 cycles of denaturation at 95 °C for 30 s, annealing at the respective Tm for each set of primers for 30 s, and extension at 72 °C for 1 min. PCR amplicons were purified with a PCR purification Kit. The PCR fragments were ligated into pGEM-T Easy vector (Promega, Madison, WI, USA). Cloned plasmids were transformed into DH5α competent cells. Transformed cells were selected using LB/ampicillin agar plates. Colonies were randomly picked to extract plasmid DNA for sequencing. During bisulfite conversion, cytosines (C) are converted into thymidines (T), but 5-methylcytosines remain unaltered. DNAMan program was used for sequence alignment and analysis. […]

Pipeline specifications

Software tools methPrimer, DNAMAN
Application BS-seq analysis
Diseases Leukemia, Neoplasms
Chemicals Zinc