Methylcap kit with high salt elution

Example 1

Methyl-binding domain (MBD)-sequencing was performed on six melanoma cell lines (WM35, WM3248, WM164, A375, M14, SK-MEL-28) and normal human epidermal melanocytes (NHEM) provided by Dr. Léon van Kempen (McGill University, Montreal, Canada). Authentication of all cell lines was performed using short tandem repeat (STR) profiling (DSMZ, Braunschweig, Germany). WM cell lines were cultured in W489 medium consisting of four parts of MCDB153 (Sigma-Aldrich, Zwijndrecht, The Netherlands) and one part of L15 (Sigma-Aldrich, Zwijndrecht, The Netherlands), A375, M14, and SK-MEL-28 cells were cultured in Dulbecco's modified Eagle's medium (Invitrogen, Breda, the Netherlands). Cells were supplemented with 2% or 10% heat inactivated fetal calf serum (Hyclone Perbio Science, Erembodegem-Aalst, Belgium), respectively. NREM cells were cultured in ready-to-use medium supplied by Promocell (Heidelberg, Germany). Genomic DNA was isolated using the PUREGENE® DNA isolation kit (Gentra systems, Minneapolis, Minn.) according to the manufacturer's instructions.

Genomic DNA of all samples was subjected to methylation-enrichment sequencing using the MethylCap kit with high-salt elution (Diagenode, Liege, Belgium) as described previously [25]. For each sample, and each methylation core, the maximum read count was used in downstream analyses.

Example 1

Methyl-binding domain (MBD)-sequencing was performed on six melanoma cell lines (WM35, WM3248, WM164, A375, M14, SK-MEL-28) and normal human epidermal melanocytes (NREM) provided by Dr. Leon van Kempen (McGill University, Montreal, Canada). Authentication of all cell lines was performed using short tandem repeat (STR) profiling (DSMZ, Braunschweig, Germany). WM cell lines were cultured in W489 medium consisting of four parts of MCDB153 (Sigma-Aldrich, Zwijndrecht, The Netherlands) and one part of L15 (Sigma-Aldrich, Zwijndrecht, The Netherlands), A375, M14, and SK-MEL-28 cells were cultured in Dulbecco's modified Eagle's medium (Invitrogen, Breda, the Netherlands). Cells were supplemented with 2% or 10% heat inactivated fetal calf serum (Hyclone Perbio Science, Erembodegem-Aalst, Belgium), respectively. NREM cells were cultured in ready-to-use medium supplied by Promocell (Heidelberg, Germany). Genomic DNA was isolated using the PUREGENE® DNA isolation kit (Gentra systems, Minneapolis, Minn.) according to the manufacturer's instructions.

Genomic DNA of all samples was subjected to methylation-enrichment sequencing using the MethylCap kit with high-salt elution (Diagenode, Liege, Belgium) as described previously [25]. For each sample, and each methylation core, the maximum read count was used in downstream analyses.