design: Nuclear mRNA-seq in the differentiation system ESC to NPC upon O-GlcNAc removal
library_construction_protocol: Fresh ESCs and NPCs were washed 3 times with PBS 1X and collected at 200 g for 5 min. 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 pellets were resuspended with 1 mL of HS Lysis Buffer (50 mM KCl, 10 mM MgSO4⋅7H2O, 5 mM HEPES, 0.05% IGEPAL, 3 mM DTT, 1X cOmplete Mini EDTA-Free protease Inhibitors (Roche)) and nuclei pelleted at 1,000 g for 5 min at 4°C. Nuclei were washed three times with 1 mL of RSB Buffer (10 mM NaCl, 10 mM tris pH 8.0, 3 mM MgCl2) and pelleted at 1,000 g for 5 min at 4°C.  Messenger RNA libraries and RNA sequencing were performed with pair-ends (PE40) sequencing mode with Illumina NextSeq 500 platform.