|Application:||ChIP-on-chip analysis, ChIP-seq analysis|
|Number of samples:||12|
|Release date:||Dec 1 2016|
|Last update date:||Dec 1 2016|
|Diseases:||Deficiency Diseases, Neural Tube Defects|
|Dataset link||Geminin promoter occupancy and Geminin-dependent histone acetylation during neuroectodermal cell specification|
For ChIP sample preparation for promoter occupancy assays, three independent biological replicates each were generated for the following sample types: (1) Geminin ChIP in ESCs (2) Geminin ChIP in ESC-derived neuroectoderm after 2.5 days of N2B27 monolayer culture, using the N18 Gmnn antibody (Santa Cruz sc-8449). (3-4) A clonal A2lox embryonic stem cell line engineered for Doxycycline-dependent Gmnn knockdown (KD) was differentiated in N2B27 medium for 2.5 days with or without 500ng Doxcycline and these samples were used for H3K9ac ChIP (antibody Abcam; Ab10812) to assess Geminin-dependent histone acetylation (H3K9ac in GemKD cells on day 2.5, three biological replicates each were generated with Gmnn knockdown (plus Dox) or under control conditions (no Dox). The A2lox mouse ES cell line (Iacovino et al., Stem Cells 2011, 29(10): 1580-88) and a clonal derivative engineered for Doxycycline-dependent Gmnn shRNA.expression (Yellajoshyula et al., PNAS 2011, 108(8): 3294-99) were routinely propagated on feeder cells (mouse embryonic fibroblast cells, Chemicon) in DMEM (Invitrogen) supplemented with 15% fetal calf serum (Hyclone), 1 mM sodium pyruvate, 1 mM Non-Essential amino acids, 1mM L-Glutamine (Invitrogen), 10−4 M 2-mercaptoethanol (Sigma) and 103 units per ml of leukemia inhibitory factor (ESGRO, Chemicon). Monolayer neural differentiation of ES cells was done as previously described (Ying and Smith, Methods in Enzymology 2003, 365: 327-41). Briefly, ES cells cultured on feeder cells were dissociated and plated at 1x105/cm2 for one day on 0.1% gelatin-coated tissue culture dishes. After 24 hours, cells were plated onto 0.1% gelatin-coated tissue culture dishes at low density (1x104 cells/cm2) in N2B27 medium and cultured for 2.5 days.
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