Selective chemical labeling of 5hmC coupled with sequencing (5hmC-seq)

For each sample, 550 ng of genomic DNA was sonicated with a Bioruptor Pico in Tris 10 mM pH 8 to obtain DNA fragments of 300 bp in average. 25 pg of 5hmC control spike-in was added to the sonicated DNA (control provided by the kit HydroxyMethyl Collector, Active Motif, ref. 55013). 50 ng of DNA was conserved at this stage to make the input library later. The remaining DNA was processed using the HydroxyMethyl Collector kit (method from Song et al. [35]) to glycosylate and biotinylate specifically the genomic 5hmC. After glycosylation and biotinylation, the DNA was purified with Ampure beads (Beckman Coulter, ref. A63881). The DNA fragments containing the biot-glu-5hmC were purified with Streptavidin beads (Active Motif, ref. 55013), eluted, purified with Ampure beads and finally eluted in 50 uL Tris pH 8. The 5hmC-seq libraries were prepared with the kit NEBNext Ultra II DNA library prep kit for Illumina (ref. E7645S) and indexed with NEBNext dual indexed primers (E7600S). The libraries were quality-checked by HS DNA Agilent BioAnalyzer (Additional file 2: Figure S1), dosed by DNA HS Qubit, pooled and submitted to the genome sequencing platform for Single-Read 50 bp Illumina HiSeq-2500 Rapid Run sequencing.