alias: GSM3375717
title: GSM3375717: Wild type line #5; Mus musculus; MeDIP-Seq
library_construction_protocol: Genomic DNA was extracted with Qiagen Dneasy Blood & Tissue kit according to the manufacturer's protocol. 100 ng genomic DNA extracted from ES cells were fragmented in 50 μL Tagmentation buffer at 55°C. Fragmented DNA was purified by Zymo DNA clean&concentrator Kit (Zymo Research, Tustin, CA). Then, the selective 5hmC chemical labeling was performed in 25 μL glucosylation buffer (50 mM HEPES buffer pH 8.0, 25 mM MgCl2) containing above fragmented DNA, 100 μM N3-UDP-Glc, 1 μM β-GT, and incubated at 37°C for 2 hr. After purified in 45 μL ddH2O, 1.5 μL DBCO-PEG4-Biotin (Click Chemistry Tools, 4.5 mM stored in DMSO) was added and incubated at 37°C for 2 hr. The biotin labeled DNA was pulled down by 5 µL C1 Streptavidin beads (Life Technologies, Carlsbad, CA) for 15 min at room temperature. Next, the captured DNA fragments were subjected to 13 cycles of PCR amplification using Nextera DNA sample preparation kit (Illumina, San Diego, CA). The resulting amplified product was purified by 1.0X AMPure XP beads. Input library was made by direct PCR from fragmented DNA without chemical labeling and capture. The libraries were quantified by a Qubit fluorometer (Life Technologies) and sequenced on an Illumina HiSEQ2500 sequencer with paired-end 40-bp reads.
identifiers.namespace: GEO
identifiers.id: GSM3375717