title: ATAC-seq in the differentiation system ESC to NPC upon O-GlcNAc removal
design: ATAC-seq in the differentiation system ESC to NPC upon O-GlcNAc removal
library_construction_protocol: Prior to harvesting, cells were treated with DNase at a final concentration of 200 U/ml for 30 min at 37°C to digest possible degraded DNA released from dead cells. Cells were washed with PBS 1X, detached with TrypLE and 50,000 cells for each condition were pelleted at 500 g for 5 min at 4°C. Differentiation of mES cells to neuronal progenitor cells (NPCs) was performed as previously described (Petracovici 2021). To obtain NPCs, ESCs were seeded in Basal Medium supplemented with 2i/LIF at a density of 20,000 cells/cm2 on gelatinized-coated plates and cultured for 12h to allow for cell attachment. Differentiation was induced by replacing the medium with Basal Medium supplemented with 10 ng/mL of recombinant human bFGF (R&D #233-FB); cells were maintained for 72h with daily medium exchange. Medium was then replaced with Basal Medium supplemented with 500 nM SAG (Sigma #566661); cells were maintained for 48h with daily medium exchange until NPCs collection. The cDNA encoding for O-GlcNAc hydrolase (Oga, Btgh84) from Bacteroides and for catalytically inactive (“dead”) Btgh84D242A (Boulard 2020) fused to Nuclear Localization Sequence (NLS) and CXXC domain were designed and engineered by cloning the cDNA of each construct in the exchange cassette vector 2loxP as previously described (Iacovino 2011). To engineer the 2Plox-3XNLS-BtGH84, 2Plox-3XNLS-BtGH84D242A, 2Plox-CXXC-BtGH84 and 2Plox-CXXC-BtGH84D242A conditional gene expression cell lines, A2loxP-Cre cells were grown in T2i on gelatin-coated plates and Cre expression was induced by exposing the cells to 1 μg/mL of Doxycycline (Sigma-Aldrich #3447) for 24 h. A2loxP-Cre cells were nucleofected using the P3 Primary Cell 4D X LONZA Kit (LONZA #V4XP-3024) with the specific cloned plasmid and exposed to 350 μg/mL G418 selection (Sigma Andrich #G8168-50ML) for 10 days. Positive clones were selected after single clone isolation and genotyping for the knock-in sequence. Cell pellet was resuspended in 50 μl of cold ATAC-Resuspension Buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2) supplemented with 0.1% IGEPAL, 0.1% Tween-20 and 0.01% Digitonin and incubated on ice for 3 min. Lysis was washed out with 1 mL of cold ATAC-Resuspension Buffer supplemented with 0.1% Tween-20 and cells pelleted at 500 g for 10 min at 4°C. Cell pellet was resuspended in 50 μl of transposition mixture (25 μl 2xTD buffer, 2.5 μl transposase (Illumina Tagment DNA Enzyme and Buffer Kit #20034197), 16.5 μl 1X PBS, 0.5 μl 1% digitonin, 0.5 μl 10% Tween 20, 5 μl H2O) and incubated at 37°C for 30 min in a thermomixer shaking at 1,000 RPM. Reaction clean-up was performed with DNA Clean and Concentrator-5 Kit (Zymo #D4014) and purified DNA was eluted in 21 μl of Elution Buffer.  The purified DNA was amplified by PCR amplification using a unique combination of dual barcoded primers P5 and P7 from the Nextera XT Index Kit (Illumina #15055293), with the Q5 Hot Start High-Fidelity polymerase (NEB #M0494S) and the following cycling conditions: 72°C for 5 min, 98°C for 30 sec, 5 cycles of 98°C for 10 sec, 63°C for 30 sec, 72°C for 1 min. 10% of the pre-amplified mixture was used to run a qPCR and determine the number of additional cycles needed to amplify the DNA. qPCR was run using SYgreen Blue Mix (PCRbio #PB20.15-05) and the previously used P5 and P7 primers in a LightCycler® (Roche) thermal cycler. After qPCR amplification, the amplification profiles were manually assessed by plotting linear Rn versus cycle based on previously optimized workflow analysis (Buenrostro et al., 2015) in order to perform an additional number of cycles equal to one-third of the maximum fluorescent intensity of the plot reported by the authors. The remaining DNA material was used to run the additional calculated cycles without the addition of other reagents. The final PCR reaction was purified using the DNA Clean and Concentrator-5 Kit (Zymo #D4014) and final DNA was eluted in 20 μl of H2O. Samples concentrations were verified using Qubit III and size selected from Nusieve 3:1 Agarose gel to isolate fragments from 150 bp to 800 bp and DNA purified using Monarch® DNA Gel Extraction Kit Protocol (NewEngland Laboratories #T1020). DNA quality was evaluated using Tape Station DNA HS D1000 Kit on a Tape Station system (Agilent 4150).