GSM1358045: MOMA#9; Homo sapiens; Bisulfite-Seq
Genomic DNA was isolated from the cell lines and clinical samples using DNeasy® Blood & Tissue Kit (QIAGEN) following manufacturer's protocol. The extracted genomic DNA was verified by agarose gel electrophoresis, and the concentration was estimated by NanoDrop (Thermo Scientific). Furthermore, BioAnalyzer 2100 DNA Chip was used to ensure the quality, quantity and integrity for the genomic DNA. Five µg genome DNA from the cell line models and the clinical samples was digested by restriction enzyme, MspI (New England BioLabs) over night at 37 °C and QIAGEN Mini Purification kit was used to purify the digested products. End repair, adding A and adaptors, where the cytosines in the paired end adaptor sequence were methylated, was performed. The ligated product was subjected to size selection in 2% agarose gel (Bio-RAD) at 100 V for 2 hours. Agarose gel bands with the inserted genomic DNA size 40 ~ 110 bp and the inserted genomic DNA size 110 ~ 220 bp were excised, so that two libraries were generated from the MOMA3, MOMA4, MOMA5, MOMA7, MOMA8 and MOMA9 (one consisting of 40 ~ 110 bp target sequences and the other of 110 ~ 220 bp target sequences). The rest clinical samples and all cell line samples were generated with a single library with inserted DNA fragments with 40-300 bp. The DNA from the excised gel pieces was recovered by QIAGEN Gel Extraction Purification Kit, followed by bisulfite treatment using ZYMO EZ DNA Methylation-Gold kit. The resulting converted DNA was amplified by PCR and purified. The RRBS libraries were performed paired-end 50 nt sequencing with HiSeq 2000 (Illumina).