title: GSM2205575: 54_1000_A; synthetic construct; Bisulfite-Seq
library_construction_protocol: Equal amounts of oligos containing the sequences (TTTAGGG)15 and (CCCTAAA)15 were mixed, heated to 98ºC for 10 minutes and allowed to cool down at room temperature for 15 minutes. Then, the annealed DNA fragments were gel purified and their quality assessed using the D1000 assay on the TapeStation 2200 (Agilent Technologies). End repair, A-tailing and adapter ligation was performed on the annealed oligos according to the Illumina TruSeq Nano kit protocol (cat # 15041759, Illumina, CA).  Adapter-ligated oligos were denatured in the presence of bisulfite (Zymo Lightning MagPrep, cat # D5046, Zymo Research, CA) at 98°C for 8 minutes and two temperatures were used for the conversion step: 54°C (manufacturer’s suggested temperature) and 40°C. Desulfonation was performed according to manufacturer’s instructions. Converted DNA was amplified using MyTaq Mix (Bioline, Taunton, MA,) and Illumina TruSeq PCR Primer Cocktail according to the following protocol: initial denaturation at 98°C for 30 s; 12 cycles of 98°C for 15 s, 60°C for 30 s, 72°C for 30 s; final extension at 72°C for 5 min. AMPure XP beads were used to clean up the final PCR reactions before sequencing.
attributes.tag: GEO Accession
attributes.value: GSM2205575