GSM5067174: Ctrl_P; Homo sapiens; OTHER
Genomic DNA from donors and patients was isolated with the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany). DNA of six leukemia cell lines (HL-60, KG-1a, TF-1, K562, U937 and SUP B15) was isolated with the NucleoSpin Tissue Kit (Macherey-Nagel, Düren, Germany). Assays for targeted amplicon sequencing were designed for four regions with AML associated CpGs. These regions comprise 14 neighboring CpGs around cg15289427, 10 CpGs around cg22797031, 15 CpGs around cg27630153, and 9 CpGs around cg19586199. Genomic DNA (500 ng) of blood samples or cell lines was bisulfite converted with the EZ DNA Methylation Kit (Zymo Research, Irvine, USA). Subsequently, the four AML-associated regions were amplified using the PyroMark PCR Kit (Qiagen) using primers with overhangs for subsequent barcoding. Subsequently, all four regions of a sample were pooled and purified using the Agencourt AMPure XP PCR Purification Kit (Beckman Coulter, Brea, CA, USA). Pooled regions were amplified with a 2nd round of PCR using the PyroMark PCR Kit and primers containing 8-bp long barcodes. Next, all samples were pooled, purified with the Select-a-Size DNA Clean & Concentrator Kit (Zymo Research), and library concentration was measured with the Qubit dsDNA BR Assay Kit (Thermo Fischer Scientific). The prepared amplicon library was denatured and diluted using the MiSeq Reagent Nano Kit v2 or MiSeq Reagent Kit v2 (Illumina, San Diego, CA, USA), according to the manufacturer's instruction. Due to low library diversity 15-20% of the PhiX Control (Illumina) was spiked-in. Pooled and barcoded samples were analyzed on the Illumina MiSeq Benchtop Sequencer (Illumina) using the 250 bp paired-end sequencing mode.