ESCLG6hGlcNAc_rep2
Mus musculus
Protocols: For each condition, 1x106 cells were collected and centrifuged at 600 g for 3 min at 4°C. 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. Cleavage under targets and release using nuclease (CUT&RUN) was performed as previously described on live nuclei (Skene 2017).  Nuclei were extracted by resuspending the cell pellet in 1 mL of Nuclear Extraction Buffer (20 mM HEPES-KOH pH 7.9, 10 mM KCl, 0.5 mM Spermidine, 0.1% Triton X-100, 20% Glycerol, 1X cOmplete Mini EDTA-Free Protease Inhibitors (Roche)). 25 μL of Concanavalin A magnetic beads (Bangs Laboratories #BP531) previously washed twice in Binding Buffer (20 mM HEPES-KOH pH 7.9, 10 mM KCl, 1 mM CaCl2) were gently added to each nuclei sample and incubated for 10 min at RT under gentle rotation. The bead-bound nuclei were isolated on a magnetic stand and beads were blocked with 1 mL of Blocking Buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM Spermidine, 0.1% BSA, 2 mM EDTA, 1X cOmplete Mini EDTA-Free Protease Inhibitors (Roche)) for 5 min at RT. The bead-bound nuclei were isolated on the magnetic stand, washed with 1 mL of Wash Buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM Spermidine, 0.1% BSA, 1X cOmplete Mini EDTA-Free Protease Inhibitors (Roche)), incubated for 5 min with 1 mL of Wash Buffer supplemented with 0.1% of Triton X-100, subsequently washed twice with Wash Buffer and resuspended in 300 μL of Antibody Buffer with 0.01% TritonX-100 supplemented with 1 μg of antibody. Samples were incubated under gentle rotation overnight at 4°C, washed twice with 1 mL of Wash Buffer and resuspended in 300 μL of Wash Buffer with 0.01% TritonX-100 supplemented with 700 ng/mL of pA/G MNase. The bead-bound nuclei were incubated for 1h at 4°C under gentle rotation and washed twice with Wash Buffer. The samples were resuspended in 50 μL of Wash Buffer and placed in iced water to pre-cool to 0°C. pA/G MNase targeted digestion was initiated by adding 2 μL of CaCl2, samples were mixed by flicking and incubated for 30 min on iced water at 0°C. Digestion reaction was stopped by adding 50 μL of 2X Stop Buffer (200 mM NaCl, 20 mM EDTA, 4 mM EGTA, 1% IGEPAL, 1 mM MnCl2). Samples were incubated at 37°C for 15 min to release the CUT&RUN fragments from the insoluble nuclear chromatin and centrifuged at 16,000 g for 5 min at 4°C. Supernatants were collected after magnetic beads separation on magnetic stand and transferred to new tubes. 2 μL of 10% SDS and 2.5 μL of 20 mg/ml of  Protein K were added and samples were incubated at 70°C for 10 min. DNA fragments were purified and size selected using SPRIselect magnetic beads (Beckman Coulter #B23318) following the manufacturer's protocol for single selection to purify fragments higher than 100 bp. DNA fragments were eluted in 30 μL of 0.1 M TE.  Samples concentrations were verified using Qubit III and libraries prepared with NEBNext Ultra II DNA Library Prep Kit for Illumina (E7645S) following the manufacturer's protocol. Libraries were size selected from Nusieve 3:1 Agarose gel to isolate fragments from 250 base pair (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).
ENA first public
2024-08-31
INSDC center name
EMBL
INSDC status
public
Submitter Id
E-MTAB-14309:ESCLG6hGlcNAc_rep2
broker name
ArrayExpress
cell_line
embryonic cell line
cell_type
mouse embryonic stem cell
collection date
not collected
common name
house mouse
geographic location (country and/or sea)
not collected
isolate
not applicable
replicate
2
sample name
E-MTAB-14309:ESCLG6hGlcNAc_rep2
scientific_name
Mus musculus