GSM3357497: KO2-D10_5hmC; Mus musculus; OTHER
Samples were harvested at day 0, 2, and 10 and DNA was harvest by treatment of DNA lysis buffer and proteinase K. DNA was purified by isopropanol perceipitation followed by 70% EtOH washes and resuspended in TE buffer. Samples were sonicated using Covaris S220 to 200-500 bp fragments. 50ug of Sonicated DNA was labeled with UDP-6-N3_Glc at a final concentration of 10uM using T4-BGt for 1 hour at 37C. UDP-labelled DNA was purified and treated with DBCO-S-S-PEG3_biotin at a final concetration of 150mM at 37C for 2 hours. Biotin labelled DNA was quantified and normalized and 5hmC capture was performed by incubation with 50uL of equilibrated Dynabeads MyOne streptavidin C1 beads for 15 minutes at room temperature. Captured DNA was eluted using 50mM of DTT for 2 hours and DNA was purified by Micro Bio-Spin5 Columns and Qiagen MinElute PCR purifition kit. Library was prepared by Nugen Ultra Low Library Systems via manufacture's protocol with 14 cycles of amplification.
GEO
GSM3357497