KEY RESOURCES TABLE
| REAGENT or RESOURCE | SOURCE | IDENTIFIER |
|---|---|---|
| Antibodies | ||
| Actin | NeoMarkers | Cat#ms-1295-p1 |
| Chromogranin A | Immunostar | Cat #20086 |
| E-Cadherin | R&D | Cat #AF648 |
| E-Cadherin | BD Transduction Lab | Cat #610182 |
| Glucagon | Zymed | Cat #18–0064 |
| Glucagon | Cell Marque | Cat #259R-15 |
| HA | Santa Cruz | Cat #sc-805 |
| HA | Roche | Cat #3F10 |
| Insulin | Dako | Cat #A0564 |
| Neurogenin 3 | R&D | Cat #AF3444 |
| Nkx2–2 | DSHB | Cat #74.5A5 |
| Pdx1 | Abcam | Cat #ab47383–100 |
| Somatostatin | Santa Cruz | Cat #sc-7819 |
| Synaptophysin | Synaptic Systems | Cat #101004 |
| Bacterial and Virus Strains | ||
| One Shot™ Stbl3™ Chemically Competent E. coli | Invitrogen | Cat #C737303 |
| One Shot™ TOP10 Chemically Competent E. coli | Invitrogen | Cat #C404003 |
| Lentiviral vectors pseudotyped with vesicular stomatitis virus (VSV) G-protein | CCHMC Viral Vector Core | N/A |
| Chemicals, Peptides, and Recombinant Proteins | ||
| recombinant human FGF4 | R&D Systems | Cat#235-F4 |
| recombinant human EGF | R&D Systems | Cat#236-EG-01M |
| recombinant human Noggin | R&D Systems | Cat#6057-NG |
| recombinant human BMP4 | R&D Systems | Cat#314-BP-050 |
| recombinant human FGF7 | R&D Systems | Cat#251-KG-050 |
| Activin A | Cell Guidance Systems | Cat#GFH6 |
| CHIR99021 | Stemgent | Cat#04–0004 |
| Y-27632 dihydrochloride | Tocris | Cat#1254 |
| Doxycycline | Sigma-Aldrich | Cat#D3447 |
| hESC-qualified Matrigel | BD Biosciences | Cat#354277 |
| mTeSR1 media | Stem Cell Technologies | Cat#5850 |
| RPMI 1640 Medium | Thermo Fisher Scientfic | Cat#11-875-093 |
| DMEM, High Glucose | Thermo Fisher Scientfic | Cat#11-965-092 |
| Advanced DMEM/F12 | Thermo Fisher Scientfic | Cat#12-634-010 |
| Growth Factor Reduced (GFR) Basement Membrane Matrix | Corning | Cat#356231 |
| Matrigel | BD Biosciences | Cat#354234 |
| Defined fetal bovine serum (dFBS) | Hyclone | Cat#SH30070.02 |
| Normal donkey serum | Jackson Immunoresearch Labs | Cat#017-000-121 |
| Odyssey® Blocking Buffer (TBS) | LI-COR | Cat#927–50000 |
| L-glutamine (100×) | Thermo Fisher Scientific | Cat#25030–081 |
| Pen/Strep (100×) | Thermo Fisher Scientific | Cat#15140–122 |
| 50× B27 supplement w/o Vitamin A | Thermo Fisher Scientific | Cat#12587–010 |
| Non-essential Amino Acids (100×) | Thermo Fisher Scientific | Cat#11140050 |
| HEPES Buffer | Thermo Fisher Scientific | Cat#15630080 |
| N2 Supplement | Thermo Fisher Scientific | Cat#17502–048 |
| Dispase | Thermo Fisher Scientific | Cat#17105–041 |
| Acutase | Thermo Fisher Scientific | Cat#A11105–01 |
| IntestiCult™ Organoid Growth Medium (Human) | Stem Cell Technologies | Cat#06010 |
| G-418 disulfate salt | Sigma-Aldrich | Cat#A1720 |
| Fluoromount-G® | SouthernBiotech | Cat#0100–01 |
| PD0325901 | Stem Cell Technologies | Cat#72182 |
| MG-132 | Selleckchem | Cat#S2619 |
| Cycloheximide | Sigma-Aldrich | Cat#C1988–1G |
| Critical Commercial Assays | ||
| Quantitect SYBR Green | Qiagen | Cat#204145 |
| QIAquick PCR Purification Kit | Qiagen | Cat#28104 |
| 8–16% Mini-PROTEAN® TGX™ Precast Protein Gels | Bio-Rad | Cat#4561104 |
| Nucleospin RNA | Macherey-Nagel | Cat#740955 |
| Lipofectamine™ 3000 Transfection Reagent | Invitrogen | Cat#L3000001 |
| SuperScript VILO cDNA synthesis kit | Invitrogen | Cat#11754250 |
| Dual-Luciferase® Reporter Assay System | Promega | Cat#E1910 |
| Lambda Protein Phosphatase | New England Biolabs | Cat#P0753S |
| LIVE/DEAD™ Fixable Violet Dead Cell Stain Kit | Invitrogen | Cat#L34963 |
| pENTR™/D-TOPO™ Cloning Kit | Invitrogen | Cat#K240020 |
| Gateway™ LR Clonase™ II Enzyme mix | Invitrogen | Cat#11791020 |
| Deposited Data | ||
| Western Blots and EMSA | This paper; Mendeley Data | https://data.mendeley.com/datasets/dj53dkzwp5/draft?a=3581f584-ea06-4cef-9991-6d6b6e6ab3c4 |
| Experimental Models: Cell Lines | ||
| Human: NEUROG3−/− H1 ES
background cells (passage 56) |
McGrath et al., 2015 | N/A |
| Human: H1 ES cells (passage 42): | CCHMC Pluripotent Stem cell core/WiCell Research Institute | NIH hESC-10–0043 |
| Oligonucleotides | ||
| Probes with high affinity E-box site from
NKX2–2 promoter: TTATTACCGCTGAACATATGGCCAATATTTTGACTTAGTGCGGGCGTGGCT |
IDT DNA | N/A |
| Probes with low affinity E-box site from
NEUROG3 promoter: CTTTGTCCGGAATCCAGCTGTGCCCTGCGGGGGAGTAGTGCGGGCGTGGCT |
IDT DNA | N/A |
| Dll1 E-box bound by E-protein homodimers
but not NEUROG3 complexes: AGAGAGCAGGTGCTGTAGTGCGGGCGTGGCT |
Castro et al., 2006 | N/A |
| 5’ HA-F_Neurog3 ACCTGCGCCGGTAGAAAGGATGTACCCATACGATGTTCCAGATTACG |
IDT DNA | N/A |
| 5’ HA-R_Neurog3 GCGCACCCGAGGGTTGAGGCGTAGCGTAATCTGGAACATCGTATGGG |
IDT DNA | N/A |
| E123X (367G-T)-F CAAGATCTAGACGCTGCGCTTCGCCCACAACTAC |
IDT DNA | N/A |
| E123X (367G-T)-R CAGCGTCTAGATCTTGGTGAGCTTCGCGTCGTC |
IDT DNA | N/A |
| E28X (82G-T)-F GAGCCTCGGAAGACTAAGTGACCTGCCCC |
IDT DNA | N/A |
| E28X (82G-T)-R GGGGCAGGTCACTTAGTCTTCCGAGGCTC |
IDT DNA | N/A |
| L135P (404T-C)-F ACTACATCTGGGCGCCGACTCAAACGCTGCG |
IDT DNA | N/A |
| L135P (404T-C)-R CGCAGCGTTTGAGTCGGCGCCCAGATGTAGT |
IDT DNA | N/A |
| Neurog3 cDNA TOPO-F CACCTGCGCCGGTAGAAAG |
IDT DNA | N/A |
| Neurog3 cDNA TOPO-R ACAGACAGGTCCTTTCACAG |
IDT DNA | N/A |
| R107S (319C-A)-F ACTGGACGCCCTGAGCGGTGTCCTGCC |
IDT DNA | N/A |
| R107S (319C-A)-R GGCAGGACACCGCTCAGGGCGTCCAGT |
IDT DNA | N/A |
| R93L (278G-T)-F GCGAGCTCAATCGAATGCACAACCTCAACTCGGC |
IDT DNA | N/A |
| R93L (278G-T)-R TCGATTGAGCTCGCGGTCGTTGGCCTTCTTTCG |
IDT DNA | N/A |
| S171fsX68 (510G-GG)-F TGGGGGGTCCCTCTACTCCCCAGTCTCCCAGGC |
IDT DNA | N/A |
| S171fsX68 (510G-GG)-R GAGGGACCCCCCAGTCCCCGGGGGAACCG |
IDT DNA | N/A |
| S183A Phosphor_null-F GCCTGGCTCCCGCCGCGTCG |
IDT DNA | N/A |
| S183A Phosphor_null-R CGGGAGCCAGGCTGCCAGCCTGG |
IDT DNA | N/A |
| S204A Phosphor_null-F TCCGCCTGCTTGGCCCCAGGCAGTCTGGCTTTCT |
IDT DNA | N/A |
| S204A Phosphor_null-R AGACTGCCTGGGGCCAAGCAGGCGGAAAAGGTG |
IDT DNA | N/A |
| S207A Phosphor_null-F TTGGCCCCAGGCGCTCTGGCTTTCTCAGATTTTCTGTGAAAG |
IDT DNA | N/A |
| S207A Phosphor_ null-R GAGAAAGCCAGAGCGCCTGGGGCCAAGCAGG |
IDT DNA | N/A |
| T120A Phosphor_null-F GCTCGCCAAGATCGAGACGCTGC |
IDT DNA | N/A |
| T120A Phosphor_null-R TCTTGGCGAGCTTCGCGTCGTCTGG |
IDT DNA | N/A |
| Chga-qRT-PCR_F TGTGTCGGAGATGACCTCAA |
IDT DNA | N/A |
| Chga-qRT-PCR_R GTCCTGGCTCTTCTGCTCTG |
IDT DNA | N/A |
| Ind-Neurog3-qRT-PCR_F AGGTCACTTCGTCTTCCGAG |
IDT DNA | N/A |
| Ind-Neurog3-qRT-PCR_R ACAGACAGGTCCTTTCACAG |
IDT DNA | N/A |
| Neurod1-qRT-PCR_F ATCAGCCCACTCTCGCTGTA |
IDT DNA | N/A |
| Neurod1-qRT-PCR_R GCCCCAGGGTTATGAGACTAT |
IDT DNA | N/A |
| Nkx2–2-qRT-PCR_F GGAGCTTGAGTCCTGAGGG |
IDT DNA | N/A |
| Nkx2–2-qRT-PCR_R TCTACGACAGCAGCGACAAC |
IDT DNA | N/A |
| PAX4-qRT-PCR_F TGTGCAGAGATGATTCCTGG |
IDT DNA | N/A |
| PAX4-qRT-PCR_R GAGGGTCTGGTTTTCCAACA |
IDT DNA | N/A |
| Negative control-ChIP-qPCR _F ATGGTTGCCACTGGGGATCT |
IDT DNA | N/A |
| Negative control-ChIP-qPCR _R TGCCAAAGCCTAGGGGAAGA |
IDT DNA | N/A |
| Nkx2–2 promoter-ChIP-qPCR_F TGTTTTCCTTGCCGGACTGA |
IDT DNA | N/A |
| Nkx2–2 promoter-ChIP-qPCR _R CCTACCATGTGGCAGCTGAA |
IDT DNA | N/A |
| Neurog3 promoter-ChIP-qPCR_F CGAGAGAGCAAACAGAGCGG |
IDT DNA | N/A |
| Neurog3 promoter-ChIP-qPCR _R GCCACCGGCCAATCAGC |
IDT DNA | N/A |
| Recombinant DNA | ||
| pINDUCER20 | Meerbrey et al., 2011 | Addgene # 44012 |
| Software and Algorithms | ||
| Imaris | Bitplane | N/A |
| NIS Elements | Nikon | N/A |
| BD FACSDiva | BD Biosciences | N/A |
| PRISMv7 graphing software | GraphPad Software | N/A |
| SPSS Statistics 24 | IBM | N/A |