MeDIP-Seq of human: HCC827 cell line in hypoxia
A library was prepared from 5 lg original gDNA and DNA was fragmented with the same parameters as the DNA isulfite protocol. End repair, <A> base addition and adaptor ligation steps were performed using Illuminas Paired-End DNA Sample Prep kit following the manufacturers instructions. Adaptor-ligated DNA was immunoprecipitated by anti-5mC, and MeDIP products were validated by qPCR using SYBR green mastermix (Applied Biosystems) and primers for positive and negative control regions supplied in the MeDIP kit (Diagenode). qPCR validation procedures consist of 95C 5 min, followed by 40 cycles 95C 15 s and 60C 1 min. MeDIP DNA was purified with ZYMO DNA Clean & Concentrator-5 column following the manufactures instructions and amplified by adaptor-mediated PCR in a final reaction volume of 50 ll which consisting the same reagents with self-prepared method. After excising amplified DNA between 220 and 320 bp on a 2% agarose gel, amplification quality and quantity were evaluated using an Agilent 2100 Analyzer and DNA 1000 chips.