Non Stressed breast tumor_rep3
For each sample, 1 ?g genomic DNA was randomly fragmented to <500 bp by sonication (Covaris S220). The fragments were treated with End Prep Enzyme Mix for end repairing, 5 Phosphorylation and dA-tailing in one reaction, followed by a T-A ligation to add Methylated adaptors to both ends. Size selection of Adaptorligated DNA was then performed using Beads, and fragments of ~410 bp (with the approximate insert size of 350 bp) were recovered. Then bisulfite conversion was performed using the related Kit. Each sample was then amplified by PCR for 10 cycles using P5 and P7 primers, with both primers carrying sequences which can anneal with flow cell to perform bridge PCR and P7 primer carrying a six-base index allowing for multiplexing. The PCR products were cleaned up using Beads, validated using an Qsep100 (Bioptic ? Taiwan? China), and quantified by Qubit3.0 Fluorometer (Invitrogen, Carlsbad, CA, USA)