design: ChIP-seq O-GlcNac in mouse ES cells
library_construction_protocol: For O-GlcNAc chromatin profiling, cells were detached with TrypLE, washed with PBS 1X and collected to a final number of 30 x106 for each replicate. Murine A9 wild type (WT) embryonic stem (ES) cell lines and the engineered ES cell lines were maintained in a humidified atmosphere at 37°C and 5% CO2 and grown on gelatin-coated plates in different media: t2i/L media consisted in NDiff (N2B27) (Takara#Y40002), titrated 2i (0.2 μM  PD0325901 and 3 μM CHIR 99021, Sigma-Aldrich), 1,000 U/ml leukaemia inhibitory factor (LIF), 1% FBS (Gibco), 1% penicillin streptavidin (Thermo Fisher Scientific #15140122); 2i/L media consisted in 50% NDiff (N2B27) (Takara #Y40002), 50% DMEM/F-12 (Gibco #11320033), 1% FBS (Gibco), 1 mM Sodium Pyruvate (Sigma-Aldrich #S8636), 0.1 mM Non-Essential AAs (Sigma-Aldrich #M7145), 2 mM L-Glutamine (Sigma-Aldrich #G7513), 1% penicillin streptavidin (Thermo Fisher Scientific #15140122), 0.1 mM β-Mercaptoethanol (Thermo Fisher Scientific #31350010), full 2i (1 μM PD0325901 and 3 μM CHIR 99021, Sigma-Aldrich), 1,000 U/mLl leukaemia inhibitory factor (LIF); DMEM (Thermo Fisher Scientific high glucose #11995065 or low glucose #11885084), 15% FBS (Gibco), 0.1 mM Non-Essential AAs (Sigma #M7145), 2 mM L-Glutamine (Sigma-Aldrich #G7513), 1% penicillin streptavidin (Thermo Fisher Scientific #15140122), 0.1 mM β-Mercaptoethanol (Thermo Fisher Scientific #31350010), 1,000 U/mLl leukaemia inhibitory factor (LIF), titrated 2i (0.2 μM PD0325901 and 3 μM CHIR 99021, Sigma-Aldrich). The mMedium was changed daily and cells were  passaged every 2-3 days via dissociation in TrypLE Express Enzyme (Thermo Fisher Scientific #12604039). Mycoplasma-free status of the cell cultures was verified periodically.  None. No specific treatment was performed for this experiment. Cells were resuspended in 45 mL PBS 1X and 3.5 mL of Fixing Solution (50 mM HEPES-KOH pH 7.5, 100 mM NaCl, 1 mM EDTA pH 8.0, 0.5 mM EGTA pH 8.0) and were fixed with 1.1% formaldehyde (Sigma-Aldrich #252549) for 10 min at RT. The fixing reaction was quenched with 125 mM Glycine and cells were washed with PBS 1X at 2,000 g for 6 min. Cells were lysed in Lysis Buffer 1 (50 mM HEPES-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA pH 8, 10% Glycerol, 0.5% IGEPAL, 0.25% Triton X-100, cOmplete Mini EDTA-Free Protease Inhibitors (Roche)), and washed in Lysis Buffer 2 (10 mM Tris-HCl pH 8, 200 mM NaCl, 1 mM EDTA pH 8, 0.5 mM EGTA pH 8, protease inhibitors). Soluble chromatin was sheared by sonication in Sonication Buffer (50 mM HEPES pH 7.5, 140 mM NaCl, 1 mM EDTA pH 8.0, 1 mM EGTA pH 8.0, 1% Triton X-100, 0.1% C24H40O4, 0.1% SDS, cOmplete Mini EDTA-Free Protease Inhibitors (Roche)) in 1 mL Covaris tubes (Covaris #520081) using a Covaris 220 system to a chromatin average size of 200-250 bp. Soluble chromatin was separated by collecting the supernatant after centrifugation at 18,000 g for 2 min at 4°C. 60 μg or 100 μg of chromatin were immunoprecipitated overnight at 4°C with 10 μg of antibody. Samples were incubated with 50 μL of Dynabeads protein G magnetic beads (Life Technologies), previously blocked with 0.5% BSA for 4h at 4°C. Bead-bound chromatin was washed once with 1 mL of Sonication Buffer, twice with 1 mL of Sonication Buffer supplemented with 500 mM NaCl, twice with 1 mL of Sonication Buffer supplemented with 1 M NaCl and once with 1 mL of LiCl Wash Buffer (20 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0, 250 mM LiCl, 0.5% NP-40, 0.5% C24H40O4). Beads-bound chromatin was eluted twice for 15 min at 65°C in Elution Buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA, 1% SDS) and de-crosslinked overnight at 65°C. Chromatin was treated with 0.2 mg/mL of RNaseA at 37°C for 1h and with 0.2 mg/mL of Proteinase K at 55°C for 30 min. DNA was isolated by phenol/chloroform extraction followed by ethanol precipitation and resuspended in 30 μL of 0.1 M TE. Libraries were prepared with NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Laboratories #E7645S) following the manufacturer's protocol. Libraries were size selected by electrophoresis on  Nusieve 3:1 agarose gel to isolate fragments from 250 bp to 460 bp and DNA purified using Monarcharch DNA Gel Extraction Kit Protocol (New England Laboratories #T1020). Libraries quality was evaluated using Tape Station DNA HS D1000 Kit on a Tape Station system (Agilent 4150).