Sonicator

For each sample, cells were trypsinized for 8–10 min, trypsin was quenched with 10 mL of ES media, and 10⁷ cells were obtained. Cells were spun down, washed with 10 mL PBS, fixed for 12 min in a mix of formaldehyde (to a final concentration of 1%) and Fix Buffer (50 mM HEPES pH 8.0, 1 mM EDTA, 0.5 mM EGTA, 100 mM NaCl), and then quenched by glycine (final concentration of 0.125 M). Cells were incubated on ice, and then spun down at 1000g for 5 min. Nuclei were prepared by consecutive washes with Rinse 1 Buffer (50 mM HEPES pH 8.0, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP40, 0.25% Triton X100), Rinse 2 Buffer (10 mM Tris pH 8.0, 1 mM EDTA, 0.5 mM EGTA, 200 mM NaCl), and Shearing Buffer (0.1% SDS, 1 mM EDTA, 10 mM Tris HCl pH 8). After a final spin down, cells were resuspended in 100 μL of Shearing Buffer and 1× protease inhibitor cocktail (Calbiochem) nanodroplets (generous gift from Samantha Pattenden),^{40 (link)} and sonicated in an E110 Covaris Sonicator for 4 min or until DNA was sheared to between 70bp and 500bp (as confirmed by agarose gel).
After sonication, the rest of the protocol was performed with a ChIP-IT High Sensitivity Kit (Active Motif, 53040), and an H3K27ac antibody was used for the pull down (Abcam, ab4729).

Genomic DNA was extracted from frozen ground-up samples and fragmented to a range between 150 and 500 bp (mean 200 bp) in size using a Covaris sonicator prior to immunoprecipitation with 5hmC (Active Motif, CA, USA: Cat no. 39769) or 5mC (Eurogentec, Seraing, Belgium: Cat no. BI-MECY-1000) antibodies. For full DNA preparation and hMeDIP and MeDIP protocols, see [19 (link)]. In brief, 5mC and 5hmC-marked DNA was enriched following antibody enrichment and purified using DNA Clean and Concentrator™ (Zymo Research, CA, USA) prior to preparation for genome-wide sequencing on the Ion Proton semiconductor sequencer.