GSM7988244: ZR-75-1 fulvestrant-resistant cultured without fulvestrant; Homo sapiens; Bisulfite-Seq
DNA extraction was performed with the DNeasy Blood & Tissue kit (Qiagen) for 4 million cells per sample. The EZ DNA Methylation-Lighting Kit (Zymo Research) was used for bisulfite conversion of 1 µg of genomic DNA per sample. PCR amplification of twelve 300-450 bp regions was done using KAPA HiFi HotStart Uracil ReadyMix Kit (Roche). PCR products were pooled per sample, resulting in 19 amplicon pools which were purified using the HighPrep PCR Clean-up System (MagBio Genomics Inc.) at a 1:1 ratio to remove fragments below 250 bp. The DNA concentration was determined using the Qubit dsDNA Broad Range Assay Kit (Invitrogen). The NEBNext Ultra II DNA Library Prep with beads and NEBNext Multiplex Oligos for Illumina (both from New England Biolabs) were used for preparation of sequencing libraries from 400 ng pooled and purified PCR products per sample. Library quality was evaluated using a Bioanalyzer with a High Sensitivity DNA chip (Agilent Technologies) and library concentration was determined with the Qubit dsDNA Broad Range Assay kit.