GSM1525023: OCI-AML5; Homo sapiens; Bisulfite-Seq
DNA extracted by Phenol:Chloroform:Isoamylalcohol (Sigma) We spiked genomic DNA (1 or 2 μg) with unmethylated λ DNA (5 ng of λ DNA per μg of genomic DNA) (Promega). We sheared DNA by sonication to 50–500 bp with a Covaris E220 and selected 150- to 300-bp fragments using AMPure XP beads (Agencourt Bioscience Corp.). We constructed genomic DNA libraries using the TruSeq Sample Preparation kit (Illumina Inc.) following Illumina’s standard protocol. After adaptor ligation, we treated DNA with sodium bisulfite using the EpiTect Bisulfite kit (Qiagen) following the manufacturer's instructions for formalin-fixed and paraffin-embedded (FFPE) tissue samples. We performed two rounds of conversion to achieve >99% conversion. We enriched adaptor-ligated DNA through seven cycles of PCR using the PfuTurboCx Hotstart DNA polymerase (Stratagene). We monitored library quality using the Agilent 2100 BioAnalyzer (Agilent) and determined the concentration of viable sequencing fragments (molecules carrying adapters at both extremities) by quantitative PCR using the Library Quantification Kit from KAPA Biosystems. We performed paired-end DNA sequencing (two reads of 100 bp each) using the Illumina Hi-Seq 2000.