KEY RESOURCES TABLE
| REAGENT or RESOURCE | SOURCE | IDENTIFIER |
|---|---|---|
| Antibodies | ||
| Anti-OCT4 | Santa Cruz | Cat#sc-5279; RRID: AB_628051 |
| Anti-TBX18 | Santa Cruz | Cat#sc-514486 |
| Anti-mCherry | Abcam | Cat#ab183628 |
| Anti-EZH2 | cell signaling | Cat#5246s |
| Anti-H3K27me3 | active motif | Cat#39155 |
| Anti-EED | EMD Millipore | Cat#09–774 |
| Anti-JARID | cell signaling | Cat#13594s |
| Anti-H3K27ac | Active motif | 39133 |
| Anti-p300 | Santa Cruz | sc-584 |
| Anti-Pol II CTD | santa cruz | sc-47701 |
| Anti-Pol II CTD PS5 | covance | MMS-134R-200 |
| Anti-p16 | thermo fisher Scientific | MA517054 |
| Anti-p21 | cell signaling | Cat#2946 |
| Anti-H3K4me3 | abcam | ab272143 |
| Anti-H3 | abcam | AB1791 |
| Anti-GATA3 | cell signaling | Cat#5852 |
| Anti-CGB | Invitrogen | Cat#PA558598 |
| Anti-HLA | Novus Biologicals Inc | Cat#NBP1–43123 |
| Anti-Actin | Cell Signaling | Cat#4970S |
| Anti-Flag | Sigma-Aldrich | Cat#F7425 |
| Anti-Cas9 | cell signaling | Ab #14697 |
| Anti 5mC | Active Motif | AB_2793653 |
| Anti 5hmC | Active Motif | AB_10013602 |
|
Bacterial and virus strains | ||
| 10-BETA Competent Cells | New England BioLabs | C3019H |
| DH5-alpha Competent Cells | New England BioLabs | C2987I |
|
Chemicals, peptides, and recombinant proteins | ||
| Matrigel | Corning | 356231 |
| Doxycycline | Millipore-Sigma | D9891–10G |
| Neurobasal | Thermo Fisher Scientific | 21103–049 |
| N2 supplement | Thermo Fisher Scientific | 17502–048 |
| B27 supplement | Thermo Fisher Scientific | 12587–010 |
| knockout serum replacement | Thermo Fisher Scientific | A3181502 |
| hLIF | SPEED Biosystems, LLC | YSP1249 |
| GS3Ki | Selleckchem | S7566 (2017), S2924 (2014), 72054 (2016) |
| Rocki Y27632 | R & D Systems (R&D) | 1254 |
| (S)-(+)-Dimethindene maleate Mino | santa Cruz Biotechnology Inc | sc-361329 |
| IWR | Selleckchem | S7086 |
| DMEM/F12 w/o HEPES or L-Glu | Invitrogen (Gibco/BRL Life Tech) | 21331–020 |
| l-ascorbic acid 2-phosphate Mg | sigma | A8960–5G |
| Insulin-Transferrin-Selenium | Fisher Scientific | 41400045 |
| NaHCO3 | sigma | S7277 |
| FGF4 | Life technologies | PHG0263 |
| TGF-beta1 | peprotech | 100–21 |
| Heparin | Stem Cell Technologies | 7980 |
| ITS-X supplement | Thermo fisher | 51500056 |
| NRG1 | Cell Signaling | 5218SC |
| A83–01 | Miltenyi Biotec Inc. | 130–105-333 |
| Forskolin | StemCell Technologies | 72112 |
| Alamar Blue | Thermo fisher Scientific | A50100 |
| Click iT EdU | ThermoFisher Scientific | C10337 |
|
Critical commercial assays | ||
| qiagen PCR purification | Quiagen | 28104 |
| qiagen gel purification | Quiagen | 28706 |
| mini-prep | Quiagen | 27104 |
| maxi-prep | Quiagen | 12662 |
| T7 maxi invitro transcription | invitrogen | AM1314 |
| Iscript | biorad | 1708891 |
| RT | Applied Biosytems | 205111 |
| RNAiMax | invitrogen | 13778030 |
| spCas9 | sigma | CAS9PROT |
| Cell Line NucleofectorTM Kit V | lonza | VCA-1003 |
| Quick Extract | Lucigen | QE09050 |
| ExoSAP-IT | Thermofisher | 78200.200.UL |
| GoTaq® DNA Polymerase | promega | M3001 |
| pGEM®-T Easy Vector Systems | promega | A1360 |
| Q5® High-Fidelity DNA Polymerase | NEB | M0491S |
|
Deposited data | ||
| RNA-seq data | GEO | GSE155022 |
| ChIP-seq data | GEO | GSE195552 |
| Cut and Run data | GEO | GSE195553 |
| ATAC-seq data | GEO | GSE195699 |
| DEseq and bulk RNAseq | This study | Data S1 |
|
Experimental models: Cell lines | ||
| WTC-11 | Coriell Institute | GM25256 |
| Elf1-iCas9 | Ferreccio et al. | RRID:CVCL_VR49 |
| IR-MEF | ISCRM hESC CORE | N/A |
| DIPG | gift from M Monje at Stanford University | N/A |
| WTC-11 EBdCas9 | This study | N/A |
| WTC-11 NCdCas9 | This study | N/A |
| WTC-11 EBdCas9 TBX18 TATAdeletion clone 11 |
This study | N/A |
| WTC-11 EBdCas9 TBX18 TATAdeletion clone 23 |
This study | N/A |
| Elf1 EBdCas9 | This study | N/A |
| Elf1 NCdCas9 | This study | N/A |
|
Oligonucleotides | ||
| See Table S5 for oligonucleotide information | N/A | |
|
Recombinant DNA | ||
| KRAB-dCas9 | Concklin Lab | Mandegar et al., 2016 |
| EB-Flag | Our Lab | Moody et al., 2017 |
| NC-Flag | Our Lab | Moody et al., 2017 |
| EBdCas9 | This paper | N/A |
| NCdCas9 | This paper | N/A |
|
Software and algorithms | ||
| Element | Element | http://lifefaculty.biu.ac.il/gershon-tamar/index.php/element-description/element |
| Fantom | FANTOM 5 | http://fantom.gsc.riken.jp/5/ |
| R | The R Foundation | https://www-r-project-org.offcampus.lib.washington.edu/ |
| ImageJ | NIH | https://imagej-nih-gov.offcampus.lib.washington.edu/ij/ |
| Microsoft office | Microsoft | https://products.office.com |
| Snapgene | Snapgene | https://www.snapgene.com |
| Transcription factor prediction Tool | http://www.ifti.org/cgi-bin/ifti/Tfsitescan.pl | |
| QGRS Mapper Tool | http://bioinformatics.ramapo.edu/QGRS/analyze.php | |
| GEO DOI | This paper | https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE195555 |