title: GSM4830118: C1_diff_day5_4_MIP20; Homo sapiens; MeDIP-Seq
library_construction_protocol: Genomic DNA was isolated from the harvested cells using the PureLink™ Genomic DNA Mini Kit (ThermoFisher Scientific, K182002) and about 3 µg DNA (except for two samples which were of 1µg) was fragmented to 100- to 500-bp fragments in a water bath sonicator (Bioruptor Pico, Diagenode) using 30 sec on/off cycles for 10 min. MBD2 enrichment was performed with the MethylMiner™ Methylated DNA Enrichment Kit (ThermoFisher Scientific, Cat# ME10025). Approximately 1-2 μg of fragmented genomic DNA was incubated with human MBD2-biotin Protein coupled to paramagnetic Dynabeads® M-280 Streptavidin for methylated CpG enrichment  according to the manufacturer's protocol and the methylated CpG fragments were finally eluted twice with high salt (2000 mM NaCl) elution buffer . The enriched DNA was purified and concentrated by ethanol precipitation and the DNA concentration was measured with Quant-iT™ dsDNA HS (High Sensitivity) Assay (ThermoFisher Scientific, Cat # Q32854). Enrichment of methylated DNA with the MBD2 pulldown was confirmed by qPCR of the methylated positive control region of BRD1 and the unmethylated negative control region of CoQ3 (Taiwo et al., Nat. Protocols, 2012 and oligo sequences provided in the table below).  Enriched DNA was then sent to GTAC sequencing core for the library preparation and sequencing. (From GTAC) MeDIP DNA was run on Agilent bioanalyzer high sensitivity chips to assay size range. 10ng of DNA as determined by Qubit was blunt ended, had addition of “A” base to 3' end, and had sequencing adapters ligated to the ends. The fragments were size selected to 200-600 base pairs and underwent amplification for 15 cycles with primers incorporating p5 and p7 sequences and a unique index tag for multiplexing. The resulting libraries were sequenced using the Illumina HiSeq3000 as paired reads extending 150 bases.