title: GSM2166065: mESC_1000cell_1_nano-hmC-Seal; Mus musculus; MeDIP-Seq
library_construction_protocol: DNA was isolated using the AllPrep DNA Mini Kit from Qiagen. The tagmentation reactions were performed in a 50μl solution with 1X tagmentation buffer from Nextera DNA sample preparation kit (FC-121-1031). The input DNA is ranged from 0.5 ng to 50 ng. The reactions were performed according to the manufacturer’s instruction. The glucosylation reactions were performed in a 20 μl solution containing 50 mM HEPES buffer (pH 8.0), 25 mM MgCl2, purified DNA, 100 μM N3-UDP-Glc, and 1 μM βGT, at 37°C for 1 hr. After that, DBCO-PEG4-DBCO (Click Chemistry Tools, 20 mM stock in DMSO) was added to the reaction mixture. The reactions were incubated at 37°C for 2 hr. Next, the DNA was purified by Micro Bio-Spin 30 Column (Bio–Rad). The purified DNA incubated with 5 µL C1 Streptavidin beads (Life Technologies) in 2X buffer (5 mM Tris pH 7.5, 0.5 mM EDTA, 1 M NaCl) for 15 min. The beads were subsequently undergone six 5-min washes each with 1X buffer. All binding and washing were done at room temperature with gentle rotation. The captured DNA fragments were amplified with 12-18 cycles of PCR amplification using the enzyme mix supplied by the Nextera kit. The PCR products were purified using AMPure XP beads. Separate input libraries were made by direct PCR from fragmented DNA without labeling and capture.
attributes.tag: GEO Accession
attributes.value: GSM2166065