KEY RESOURCES TABLE
| REAGENT or RESOURCE | SOURCE | IDENTIFIER |
|---|---|---|
| Antibodies | ||
| FITC anti-human CD34 | BioLegend | Cat# 343604; RRID:AB_1732005 |
| PE/Cy7 anti-human CXCR4 | BioLegend | Cat# 306514; RRID:AB_2089651 |
| Goat anti-human/mouse/rat p53 | R&D Systems | Cat# AF1355-SP; RRID:AB_354749 |
| Rabbit anti-human c-Myc | Cell Signaling | Cat# 5605S; RRID:AB_1903938 |
| Donkey anti-goat IgG PE | Thermo Fisher Scientific | Cat# PA1-29953; RRID:AB_10983834 |
| Brilliant Violet 421 donkey anti-rabbit IgG | BioLegend | Cat# 406410; RRID:AB_10897810 |
| PE-Cy7 rat anti-mouse Ly-6A/E | BD Biosciences | Cat# 558162; RRID:AB_647253 |
| APC anti-mouse CD117 (c-kit) (Clone ACK2) | BD Biosciences | Cat# 135108; RRID:AB_2028407 |
| Pacific Blue anti-mouse CD150 (Clone TC15-12F12.2) | BioLegend | Cat# 115924; RRID:AB_2270307 |
| APC-Cy7 anti-mouse CD48 (Clone HM48-1) | e-Bioscience | Cat# 47-0481-82; RRID:AB_2573962 |
| Rabbit anti-human phospho-KAP1 (S824) | Abcam | Cat# ab70369; RRID:AB_1209417 |
| Rabbit anti-human/mouse KAP1 | Abcam | Cat# ab10484; RRID:AB_297223 |
| Rabbit anti-human/mouse/rat/monkey phospho-Chk1 (Ser345) (133D3) | Cell Signaling | Cat# 2348S; RRID:AB_331212 |
| Anti-human/mouse/rat CHK1 (G-4) | Santa Cruz | Cat# sc-8408; RRID:AB_627257) |
| Rabbit anti-human phospho RPA32 (S33) | Bethyl Laboratories | Cat# A300-246A-M; RRID:AB_2779098 |
| Rabbit anti-human/mouse RPA32 | Bethyl Laboratories | Cat# A300-244A-M; RRID:AB_2779096 |
| Rabbit anti-human/mouse phospho-Histone H2A.X (Ser139) (20E3) | Cell Signaling | Cat# 9718S; RRID:AB_2118009 |
| Mouse anti-human vinculin (H-10) | Santa Cruz | Cat# sc-25336; RRID:AB_628438 |
| PE Donkey anti-rabbit IgG | BioLegend | Cat# 406421; RRID:AB_2563484 |
| Rat anti-BrdU (clone BU1/75 (ICR1) | Abcam | Cat# ab6326; RRID:AB_305426 |
| Mouse anti-BrdU | BD Biosciences | Cat# 347580; RRID:AB_400326 |
| Anti-mouse IgG, HRP-linked | Cell Signaling | Cat# 7076S; RRID:AB_330924 |
| Anti-rabbit IgG, HRP-linked | Cell Signaling | Cat# 7074S; RRID:AB_2099233 |
| APC anti-human CD34 (Clone 561) | BioLegend | Cat# 343608; RRID:AB_2228972 |
| PE anti-human CD19 (Clone 4G7) | BioLegend | Cat# 392506; RRID:AB_2750097 |
| PE anti-human CD11b (Clone ICRF44) | BioLegend | Cat# 301306; RRID:AB_314158 |
| PE anti-human CD14 (Clone 63D3) | BioLegend | Cat# 367103; RRID:AB_2565887 |
| PE anti-human CD56 (NCAM) (Clone 5.1H11) | BioLegend | Cat# 362508; RRID:AB_2563925 |
| PE anti-human CD235a (Glycophorin A) (Clone HI264) | BioLegend | Cat# 349106 RRID:AB_10640739 |
| Bacterial and Virus Strains | ||
| Biological Samples | ||
| Human whole bone marrow and blood from patients with FA | Boston Children’s Hospital (USA) and Instituto Nacional de Pediatria (Mexico) | N/A |
| Human whole bone marrow and blood from healthy donors | Lonza | Cat# 1M-105 |
| Human CD34+ UCB (single donor) | Cincinnati Children’s Translational Core Cell Processing Core | https://www.cincinnatichildrens.org/research/cores/translational-core-laboratory/cell-processing-core |
| Human CD34+ UCB (single donor) | StemCell Technologies | Cat# 70008.5 |
| Chemicals, Peptides, and Recombinant Proteins | ||
| UM171 | Selleck Chemicals | Cat# S7608; CAS:1448724-09-1 |
| FLT3L | Peprotech; StemCell Technologies | Cat# 300-19; Cat# 78009 |
| SCF | Peprotech; StemCell Technologies | Cat# 300-07; Cat# 78062 |
| TPO | Peprotech; StemCell Technologies | Cat# 300-18; Cat# 78210 |
| IL-6 | Peprotech; StemCell Technologies | Cat# 200-06; Cat# 78050 |
| TNF-α | Peprotech | Cat# 300-01A |
| IFN-γ | Peprotech | Cat# 300-02 |
| (+)-JQ1 | Apex Bio; Selleck Chemicals | Cat# A1910; Cat# S7110. CAS:1268524-70-4 |
| IdU | Sigma-Aldrich | Cat# I7125; CAS:54-42-2 |
| CldU | Sigma-Aldrich | Cat# C6891; CAS:50-90-8 |
| Crystal violet | Sigma-Aldrich | Cat# C0775; CAS:548-62-9 |
| Mitomycin C | Sigma-Aldrich | Cat# M0503; CAS:50-07-7 |
| PMSF | Cell Signaling | Cat# 8553S; CAS:329-98-6 |
| Alt-R S. pyogenes Cas9 nuclease V3 | Integrated DNA Technologies, Inc. | Cat# 1081059 |
| Platinum SuperFi DNA polymerase | Thermo Fisher Scientific | Cat# 12351010 |
| Platinum SuperFi II DNA polymerase | Thermo Fisher Scientific | Cat# 12361050 |
| ExoSAP-IT Express | Thermo Fisher Scientific | Cat# 75001 |
| Critical Commercial Assays | ||
| Human Cytokine Array Proinflammatory Focused 13-plex (HDF13) | Eve Technologies Corporation | https://www.evetechnologies.com/multiplex-assay/ |
| Mouse Cytokine Array Proinflammatory Focused 10-plex (MDF10) | Eve Technologies Corporation | https://www.evetechnologies.com/multiplex-assay/ |
| Deposited Data | ||
| Single-cell RNA-seq of primary human hematopoietic stem and progenitor cells from patients with Fanconi anemia and healthy donors | This paper | GEO: GSE157591 |
| Experimental Models: Cell Lines | ||
| EUFA316 +EV cell line | Zhang et al., 2016 | N/A |
| EUFA316 +G cell line | Zhang et al., 2016 | N/A |
| Mouse derived MS-5 stromal cell line | Dr. Paul Kincade, Oklahoma Medical Research Foundation | N/A |
| Mouse derived OP9-DL4 stromal cell line | Dr. Paul Kincade, Oklahoma Medical Research Foundation | N/A |
| Experimental Models: Organisms/Strains | ||
| Fancd2−/− mice C57BL/6 background | Parmar et al., 2010 | N/A |
| Oligonucleotides | ||
| Guidestrands for FANCA, FANCG and MYC targeting, see Supplementary Table S4 | This paper | N/A |
| Primers for PCR amplification and sequencing of targeted FANCA, FANCG and MYC, see Supplementary Table S5 | This paper | N/A |
| Recombinant DNA | ||
| pMD2.G plasmid | Addgene | Cat# 12259 |
| psPAX2 plasmid | Addgene | Cat# 12260 |
| pLKO.1 cloning vector | Addgene | Cat# 10878 |
| Software and Algorithms | ||
| Synthego ICE | Synthego | https://ice.synthego.com |
| Cell Ranger (version 2.1.1) | https://support.10xgenomics.com/singlecell-geneexpression/software/overview/welcome | |
| R package Seurat (version 2.3.4) | Butler et al., 2018 | https://satijalab.org/seurat/ |
| R version 3.6 | https://www.r-project.org/ | |
| R package AUCell (version 1.6.0) | Aibar et al., 2017 | |
| Molecular Signatures Database (version 5) | https://www.gsea-msigdb.org/gsea/msigdb/index.jsp | |
| R package STEMNET (version 0.1) | Velten et al., 2017 | |
| FlowJo, version 10.5.3 | https://www.flowio.com/ | |
| OpenComet plugin | http://www.cometbio.org/ | |
| Image J | http://fiji.sc | |
| Graphpad Prism 8 | Graphpad | https://www.graphpad.com/scientificsoftware/prism/ |
| Other | ||
| P3 Primary Cell 4D Nucleofector Kit | Lonza | Cat# V4XP-3024 & V4XP-3032 |
| PureLink PCR purification kit | Thermo Fisher Scientific | Cat# K310001 |
| EasySep Kit | StemCell Technologies | Cat# 19356 |
| EasySep buffer | StemCell Technologies | Cat# 20144 |
| EasySep magnet | StemCell Technologies | Cat# 18000 |
| MACS MultiStand | Miltenyi | Cat# 130-042-303 |
| CD34 MicroBead Kit, human | Miltenyi | Cat# 130-046-702 |
| FcR Blocking Reagent, human | Miltenyi | Cat# 130-059-901 |
| Human LDL | StemCell Technologies | Cat# 02698 |
| Stemspan SFEM II | StemCell Technologies | Cat# 09655 |
| MethoCult H4434 Classic | StemCell Technologies | Cat# 04434 |
| Methocult GF M3434 methylcellulose | StemCell Technologies | Cat# 03444 |
| Ammonium chloride solution | StemCell Technologies | Cat# 07800 |
| CytoFix/CytoPerm | BD Biosciences | Cat# 554714 |
| PermWash | BD Biosciences | Cat# 554723 |
| Transcription Factor Fix/Perm buffer | BD Biosciences | Cat# 562574 |
| DRAQ5 | Thermo Fisher Scientific | Cat# 62251 |
| Polybrene Infection/Transfection Reagent | Sigma-Aldrich | Cat# TR-1003-G |
| Puromycin Dihydrochloride Antibiotic Solution | Mirus Bio | Cat# MIR 5940 |
| RIPA cell lysis buffer | Cell Signaling | Cat# 9803 |
| SYBR-Green dye | Invitrogen | Cat# S7567 |
| Proteinase K | Fisher Scientific | Cat# E0491 |
| Agarase | BioLab | Cat# M0392L |
| FBS | Sigma-Aldrich | Cat# F2442 |
| Hanks balanced salt solution | Lonza | Cat# 10-547F |
| HEPES | Fisher Scientific | Cat# BP299-100 |
| Penicillin-streptomycin | Gibco | Cat# 15140-122 |
| L-Glutamine | Gibco | Cat# 25030081 |
| 70 μm cell strainer | Fisher Scientific | Cat# 087712 |
| 500 mL Bottle Top Vacuum Filter, 0.45 μm | Corning | Cat# 430514 |
| Lineage cell depletion kit | Miltenyi | Cat# 130-090-858 |
| RNeasy Micro kit | Qiagen | Cat# 74034 |
| RNeasy Mini kit | Qiagen | Cat# 74134 |
| RT2 PreAMP cDNA synthesis kit | Qiagen | Cat# 330451 |
| RT2 PreAMP Pathway primer Mix | Qiagen | Cat# 330241 PBH-177Z (human) PBM-177Z (mouse) |
| qPCR Master Mix RT2 SYBR Green | Qiagen | Cat# 330533 |
| RT2 Profiler MYC Targets PCR Array | Qiagen | Cat# 330231 PAHS-177ZC (human) PAMM-177ZC (mouse) |
| Cell Titer-Glo Luminescent Cell Viability Assay | Promega | Cat# G7573 |
| MEM α | Gibco | Cat# 12571048 |
| RPMI 1640 medium | Gibco | Cat# 11875119 |
| IMDM | Gibco | Cat# 12440053 |
| CometAssay kit | Trevigen | Cat# 4250-050-K |
| 10x Chromium Controller | 10X Technologies | https://www.evetechnologies.com/multiplex-assay/ |
| Chromium Single Cell 3’ Library & Gel Bead Kit v2 | 10X Technologies | Cat# PN-120237 |
| Chromium Single Cell A Chip Kit | 10X Technologies | Cat# PN-120236 |
| Chromium i7 Multiplex Kit | 10X Technologies | Cat# PN-120262 |
| NextSeq 500/550 High Output 75 Cycle kit v2.5 | Illumina | Cat# 20024906 |