Name: GSM6944689
Instrument: Illumina HiSeq 2000
Strategy: ChIP-Seq
Source: GENOMIC
Selection: ChIP
Layout: PAIRED
Construction protocol: In brief, 5mg of genomic DNA was sonicated to 300-400 base pairs and 5hmC containing fragments were glucosylated (T4 phage ß-glucosyltransferase enzyme and UDP-6-N3-glucose). The glucosylated fragments were purified and then biotinylated (disulfide biotin linker) and pulled down with Dynabeads MyOne Streptavidin C1 beads. The 5hmC fragments were released from the beads using dithiothreitol and purified for a final time. DNA fragments were eluted in nuclease-free water and quantified by Qubit. 5mC: 3mg of genomic DNA was sonicated to 300-400 base pairs using a Covaris focused ultrasonicator. The DNA fragments were then subjected to end repair, A-tailing, adaptor ligation and USER digestion using the NEBNext Ultra II DNA Library Prep kit for Illumina (New England BioLabs, E7645S) according to the manufacturer's protocol. Following USER digestion and purification, DNA was denatured for 10 minutes at 95°C and immunoprecipitated overnight at 4°C with 4mL of either 5mC antibody (Active Motif, 39649) or IgG antibody (Sigma 12-371) in IP buffer (500mM Tris-HCl, pH 7.4, 750mM NaCl and 0.25% TritonX). The mixture was then incubated with Protein G coated Dynabeads for at least 2 hours at 4°C, washed with ice cold IP buffer and finally washed in ice cold high salt (300mM NaCl) IP buffer. After the final washing, the beads were treated with 200mL digest buffer (1X TE Buffer, pH 7.4, 0.25% SDS, 0.25% Proteinase K (2.5mg/mL)) and shaken at 1000rpm for 2 hours at 55°C. The methylated DNA was recovered by phenol:chloform:isoamyl alcohol (25:24:1) extraction followed by precipitation in 3X volume of 100% ethanol supplemented with 3mL glycogen(5mg/mL) and 15mL NaAC pH 5.2 overnight at -20°C. The next day, the DNA was pelleted and washed with 75% ethanol and dissolved in nuclease-free water. Illumina indexes were added, PCR enriched and purified using the NEBNext Ultra II DNA Library Prep kit for Illumina following the manufacturer's protocol. The NEBNext Ultra II DNA Library Prep kit for Illumina (New England BioLabs, E7645S) was used per the manufacturer's protocol for enriched and unenriched genomic DNA. An Agilent 2100 Bioanalyzer was used to confirm purity and fragmentation size of the final libraries. Libraries were sequenced pair-end (150bp) on an Illumina HiSeq platform by Admera Health, LLC.