GSM6833280: R10-A; Homo sapiens; Bisulfite-Seq
Total RNA was extracted from frozen specimens of 34 breast tumors using the mirVana miRNA isolation kit (ThermoFisher Scientific). Human total RNA (Human XpressRef Universal Total RNA, Qiagen) was used as normal reference samples for analysis. RNA library construction was performed with the TruSeq Stranded mRNA kit (Illumina) with some modifications. In particular, 100 ng of each beads-purified aqueous RNA samples were submitted to a second round of RNACleanXP beads purification to substitute water with the FPF (Fragment, Prime, Finish Mix) solution included in the library kit. Then the manufacturer's protocol was followed. The Unique-dual indexes (Illumina) were used in the adapters ligation step. Each individual library was then quantified and quality-controlled using Qubit Fluorometer (ThermoFisher Scientific) and LabChip GX (Perkin Elmer). An unbalanced pooling design was used to achieve a ratio of 1 to 8 between untreated and BS-treated samples. After libraries unbalanced pooling, the final pool was quality checked again with Qubit, LabChip GX, and qPCR (KAPA and BIORAD).