Computational protocol: SOX11 expression correlates to promoter methylation and regulates tumor growth in hematopoietic malignancies

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Protocol publication

[…] MethPrimer http://www.urogene.org/methprimer/index1.html[] was used to analyze the 2000 bp region directly upstream of the SOX11 transcription start site (the SOX11 promoter region) for the presence of CpG islands. Using the MethPrimer default algorithm, three CpG islands were identified as >200 bp regions with G and C contents >50% and Observed/Expected CpG-rates of >0.6. One additional CpG island was detected when the region size constraint was lowered to 100 bp without altering the other criteria (Figure ). The methylation status of the 5'-promoter region was determined by sodium bisulfite sequencing [] of the 213 bp CpG island located -435 to -222 bp upstream of the SOX11 transcription start site. Briefly, total genomic DNA was extracted from five million cells per cell line or primary samples, using QIAamp DNA MINI kit (QIAgen) according to the protocol of the manufacturer. To convert unmethylated cytosine to uracil, we performed bisulfite conversion of 0.5 - 1 μg of DNA with EpiTect Bisulfite Kit (QIAgen). The CpG island was amplified from bisulfite converted DNA, using primers 5'-AGA GAG ATT TTA ATT TTT TGT AGA AGG A-3'and 5'-CCC CCT TCC AAA CTA CAC AC-3' and Platinum Taq DNA polymerase (Invitrogen). PCR products were both directly sequenced as well as ligated into the vector pCR.21-TOPO (Invitrogen) for clonal analysis. Sequencing was performed by Eurofins MWG Operon (Ebersberg, Germany and GATC Biotech (Konstanz, Germany). Quality control of methylation data was performed in a standardized manner, using the BiQ Analyzer software [], http://biq-analyzer.bioinf.mpi-inf.mpg.de/index.php. Images of CpG methylation for figures -C were constructed using the BDPC web server [], using output files from BiQ Analyzer. All amplicons included in the study had, (i) bisulfite conversion rates above 95% for unmethylated non-CpG C:s to T:s, and (ii) sequence similarity above 90% compared to the original genomic sequence. […]

Pipeline specifications

Software tools methPrimer, BDPC
Application BS-seq analysis
Diseases Lymphoproliferative Disorders, Neoplasms, Hematologic Neoplasms