Table 1. Key resources: All used materials including primer and antibodies.
| Reagent or Resource | Source | Identifier |
|---|---|---|
| Antibodies | ||
| Goat Anti-Mouse IgM-UNLB | Southern Biotech | 1021–01 |
| Goat Anti-Mouse IgM-HRP | Southern Biotech | 1021–05 |
| Lyophilized Rabbit Polyclonal Antibody Ki67 Antigen | Novocastra | NCL-Ki67p |
| Cleaved Caspase-3 (Asp175) (5A1E) Rabbit mAb | Cell signaling | 9664P |
| Phospho-Histone H3 (Ser10) Antibody | Cell signaling | 9701S |
| Purified anti-Pax-5 Antibody | Biolegend | 649702 |
| Cy3-AffiniPure Donkey Anti-Rabbit IgG (H+L) | Jackson ImmunoResearch | 711-165-152 |
| Cy3 AffiniPure Donkey Anti-Rat IgG (H+L) | Jackson ImmunoResearch | 712-165-150 |
| Alexa Fluor 594 anti-mouse/human CD45R/B220 [RA3-6B2] | Biolegend | 103254 |
| Anti-Metallophilic Macrophages antibody [MOMA-1] | Abcam | ab51814 |
| Alexa Fluor 488-AffiniPure F(ab')2 Fragment Donkey Anti-Rabbit IgG (H+L) | Jackson ImmunoResearch | 711-546-152 |
| Alexa Fluor 647 anti-mouse IFN-γ [XMG1.2] | Biolegend | 505816 |
| Biotin anti-mouse IL-5 [TRFK4] | Biolegend | 504401 |
| Alexa Fluor 488 anti-mouse IL-17A [TC11-18H10.1] | Biolegend | 506909 |
| Alexa Fluor 647 anti-mouse CD3ε Antibody | Biolegend | 152319 |
| Anti-Cytokeratin 8+18 antibody (ab194130) | Abcam | ab194130 |
| Donkey anti-Guinea Pig IgG, Alexa -2788875 | Millipore Sigma | AP193SA6 |
| Mouse NKp46/NCR1 Antibody | R&D Systems | AF2225-SP |
| Alexa Fluor 647 AffiniPure F(ab')2 Fragment Donkey Anti-Goat IgG (H+L) | Jackson ImmunoResearch | 705-606-147 |
| anti-mouse/human CD45R/B220 [RA3-6B2] | Biolegend | 103206 |
| anti-mouse CD23 [B3B4] | Biolegend | 103222 |
| anti-mouse CD21/CD35 (CR2/CR1) [7E9] | Biolegend | 123416 |
| anti-mouse CD93 (AA4.1) | Biolegend | 136505 |
| anti-mouse IgM [RMM-1] 50 μg | Biolegend | 406505 |
| anti-mouse IgD [11-26c.2a] | Biolegend | 405723 |
| anti-mouse CD45.1 [A20] | Biolegend | 110706 |
| anti-mouse CD45.2 [104] | Biolegend | 109814 |
| Anti-Mouse CD3e Clone 145-2C11 | BD Biosciences | 553063 |
| anti-mouse Ly-6G [1A8] | Biolegend | 127615 |
| anti-mouse/human CD11b [M1/70] | Biolegend | 101216 |
| anti-mouse/human CD45R/B220 | Biolegend | 103227 |
| anti-mouse TCR β chain [H57-597] | Biolegend | 109208 |
| APC/Cy7 anti-mouse CD45R/B220 | Biolegend | 103224 |
| anti-mouse CD5 [53–7.3] | Biolegend | 100626 |
| Rat Anti-Mouse CD19 | Biolegend | bdb563557 |
| CD4 Monoclonal Antibody (RM4-5), PerCP-Cyanine5.5 | eBiosciences | 45-0042-82 |
| CD8a Monoclonal Antibody (53–6.7), FITC | eBiosciences | 11-0081-82 |
| CD8a Monoclonal Antibody (53–6.7), PE-Cyanine7 | eBiosciences | 25-0081-82 |
| TCR beta Monoclonal Antibody (H57-597), APC-eFluor 780 | eBiosciences | 47-5961-82 |
| APC anti-mouse CD25 Antibody (clone PC61) | Biolegend | 102011 |
| CD4 Monoclonal Antibody (GK1.5), PE | eBiosciences | 12-0041-82 |
| PE/Cy7 anti-mouse CD117 (c-Kit) Antibody | Biolegend | 105814 |
| CD3e Monoclonal Antibody (145-2C11), FITC | eBiosciences | 11-0031-82 |
| CD19 Monoclonal Antibody (eBio1D3 (1D3)), PE | eBiosciences | 12-0193-82 |
| Notch2 (D76A6) XP | Cell signaling | 5732 |
| monoclonal anti-b-actin (AC-15) | Sigma-Aldrich | A5441 |
| ECL Rabbit IgG, HRP-linked whole AB (from Donkey) | GE Healthcare | NA934 |
| ECL Mouse IgG, HRP-linked whole AB (from Donkey) | GE Healthcare | NA931 |
| Bacterial and Virus Strains | ||
| BL21 (DE3) Tuner Competent cells | Novagen | 70623 |
| Biological Samples | ||
| Chemicals, Peptides, and Recombinant Proteins | ||
| XbaI | NEB | R0145 |
| BglII | NEB | R0144 |
| Mouse IgM-UNLB | Southern Biotech | 0101–01 |
| DyLight649(Cy5)-conjugated Streptavidin | Jackson Immunoresearch | 016-490-084 |
| Dextran sulfate sodium salt (DSS, M.W. approximately 36,000–50,000, colitis grade) | MP Biomedicals | 0216011050 |
| Red blood cell lysis buffer (10x) | BioLegend | 420301 |
| BSA | Fisher Scientific | Bp1600-100 |
| FBS | HyClone | AE24573269 |
| FBS | Sigma-Aldrich | F2442 |
| Nunc MaxiSorp flat bottom | Thermo Fisher Scientific | 44-2404-21 |
| TMB substrate solution | Thermo Fisher | N301 |
| RPMI-1640 | Sigma-Aldrich | R0883 |
| Gentamicin | Gibco B.R.L | 15750–060 |
| β-mercaptoethanol | Sigma-Aldrich | M7522 |
| U-bottom 96-well | Thermo Scientific | 174925 |
| Superfrost Plus microscope slides | Fisher Scientific | 12-550-15 |
| Xylene | Fisher Scientific | X3P |
| Alcian Blue 8GX | Sigma-Aldrich | A3157 |
| Nuclear Fast Red | Vector/Sigma-Aldrich | H-3403/N3020 |
| Trilogy | Cell Marque | 920P-10 |
| Normal donkey serum | Jackson ImmunoResearch | 017-000-121 |
| Prolong Gold Antifade Reagent | Invitrogen | P36930 |
| Complete Protease Inhibitor Cocktail | Roche | 11697498001 |
| Red Blood cell lysis buffer | BioLegend | 420301 |
| ACK lysis buffer | Lonza | 10-548E |
| Cell strainer (40 μm) | Falcon | 08-771-1 |
| Cell strainer (70 μm) | Falcon | 08-771-2 |
| Critical Commercial Assays | ||
| Supersignal West Femto Chemiluminescent substrate kit | Thermo Scientific/Fisher Scientific | P134095 |
| PureLink RNA Mini kit | Life Technologies (Invitrogen) | 12183025 |
| Superscript II reverse transcriptase | Invitrogen | 18064014 |
| iTaq Universal SYBR Green Supermix | Bio-Rad | 1725121 |
| Zombie VioletTM Fixable Viability kit | BioLegend | 423113 |
| Micro RNAeasy kit | Qiagen | 74106 |
| Zymo DNA Clean and Concentrator kit | Zymoresearch | D4029 |
| Deposited Data | ||
| SplitDam data | [45] | GEO: GSE70402 |
| ATAC-Seq data | This paper | GEO: GSE149992 |
| Experimental Models: Cell Lines | ||
| mK4 cells | [93] | N/A |
| Experimental Models: Organisms/Strains | ||
| N1RA | This paper | N/A |
| N2RA | This paper | N/A |
| N1 deletion | [94] | Notch1tm1Con |
| N2 lacZ | [95] | N/A |
| C57BL/6 | Charles River | 027 |
| FVB/N | Harlan | N/A |
| B6D2F2 | Harlan | N/A |
| Oligonucleotides | ||
| N1-deletion fwd: ACGATATCGTGGTGCATACGCTCCTGTGCG | [94] | N/A |
| N1-deletion rev: GTCAGTTTCATAGCCTGAAGAACG | [94] | N/A |
| N1RA fwd: GCGGGATTCCTAGCCTGGTTACTC | This paper | N/A |
| N1RA rev: GTCCTTGTTGGCTCCGTTCTTCAG | This paper | N/A |
| N2-LacZ fwd: GTTGCAGTGCACGGCAGATACACTTGCTGA | [95] | N/A |
| N2-lacZ rev: GCCACTGGTGTGGGCCATAATTCAATTCGC | [95] | N/A |
| N2RA fwd: ACGGCTCATCCCTGACATGAG | This paper | N/A |
| N2RA rev: TCCTCTCAGAAGGGTAGCAAGTC | This paper | N/A |
| Actb qPCR fwd: GGCTGTATTCCCCTCCATCG | Harvard PrimerBank | 6671509a1 |
| Actb qPCR rev: CCAGTTGGTAACAATGCCATGT | Harvard PrimerBank | 6671509a1 |
| Fos qPCR fwd: CGGGTTTCAACGCCGACTA | Harvard PrimerBank | 6753894a1 |
| Fos qPCR rev: TTGGCACTAGAGACGGACAGA | Harvard PrimerBank | 6753894a1 |
| Gapdh qPCR fwd: AGGTCGGTGTGAACGGATTTG | Harvard PrimerBank | 6679937a1 |
| Gapdh qPCR rev: TGTAGACCATGTAGTTGAGGTCA | Harvard PrimerBank | 6679937a1 |
| Myb qPCR fwd: AGACCCCGACACAGCATCTA | Harvard PrimerBank | 19526459a1 |
| Myb qPCR rev: CAGCAGCCCATCGTAGTCAT | Harvard PrimerBank | 19526459a1 |
| Notch1 qPCR fwd: GATGGCCTCAATGGGTACAAG | Harvard PrimerBank | 13177625a1 |
| Notch1 qPCR rev: TCGTTGTTGTTGATGTCACAGT | Harvard PrimerBank | 13177625a1 |
| Notch2 qPCR fwd: ATGTGGACGAGTGTCTGTTGC | Harvard PrimerBank | 33859592a1 |
| Notch2 qPCR rev: GGAAGCATAGGCACAGTCATC | Harvard PrimerBank | 33859592a1 |
| Pax5 qPCR fwd: CCATCAGGACAGGACATGGAG | Harvard PrimerBank | 6679213a1 |
| Pax5 qPCR rev: GGCAAGTTCCACTATCCTTTG | Harvard PrimerBank | 6679213a1 |
| IL1b-qPCR-fwd: CAACCAACAAGTGATATTCTCCATG | [96] | N/A |
| IL1b-qPCR-rev: GATCCACACTCTCCAGCTGCA | [96] | N/A |
| IL4-qPCR-fwd: AGATGGATGTGCCAAACGTCCTCA | [97] | N/A |
| IL4-qPCR-rev: AATATGCGAAGCACCTTGGAAGCC | [97] | N/A |
| Claudin4-qPCR-fwd: GTCCTGGGAATCTCCTTGGC | Harvard PrimerBank | 6753440a1 |
| Claudin4-qPCR-rev: TCTGTGCCGTGACGATGTTG | Harvard PrimerBank | 6753440a1 |
| ZO1-qPCR-fwd: GCACCATGCCTAAAGCTGTC | [98] | N/A |
| ZO1-qPCR-rev: ACTCAACACACCACCATTGC | [98] | N/A |
| TNFa-qPCR-fwd: CCCTCACACTCAGATCATCTTCT | Harvard PrimerBank | 7305585a1 |
| TNFa-qPCR-fwd: GCTACGACGTGGGCTACAG | Harvard PrimerBank | 7305585a1 |
| IL17A-qPCR-fwd: GACGCGCAAACATGAGTCC |
This paper | N/A |
| IL17A-qPCR-rev: TTTGAGGGATGATCGCTGCT | This paper | N/A |
| N1RA sgRNA fwd: caccgCATTCGGGCATCCAGATCTG | This paper | N/A |
| N1RA sgRNA rev: aaacCAGATCTGGATGCCCGAATGc | This paper | N/A |
| N2RA sgRNA fwd: caccGGCATCCAGATCGGTTACA | This paper | N/A |
| N2RA sgRNA rev: aaacTGTAACCGATCTGGATGCC | This paper | N/A |
| N1RA donor oligo: g*c*t*tgcatttagatcaccctgcctgaacccatccctgccttccagatcctgctccggaacgcagctactgatctagatgcccgaatgcatgatggcacaactccactgatcctggctgcgcgcctggccgtg*g*a*g | This paper | N/A |
| N2RA donor oligo: a*g*c*caggcgggcagccaggatcaggggggtagtaccatcgttcattctggcatccagatctgttaccgcgttgcggatcagaatctagaagagaagcagagaagtgtcccttagataaggaaaaaga*a*t*g | This paper | N/A |
| 2xCSL(EMSA): cgaaCGTGGGAAacctaggctagaggcacCGTGGGAAactagtgcgggcgtggct | This paper | N/A |
| 1xSPS(EMSA): gctaCGTGGGAAaggagcaaactgcgtTTCCCACGttcgtagtgcgggcgtggct | This paper | N/A |
| 5’IRDye-700_complementary_oligo(EMSA): agccacgcccgcact | This paper | N/A |
| 5’IRDye-800_complementary_oligo(EMSA): agccacgcccgcact | This paper | N/A |
| Recombinant DNA | ||
| Plasmid: N1ΔE | [30] | N/A |
| Plasmid: N1RAΔE | This paper | N/A |
| pSpCas9(BB) | [99] | 48139 |
| pTXB1-Tn5 | [92] | N/A |
| Software and Algorithms | ||
| Image lab | Bio-Rad Laboratories | https://www.bio-rad.com/en-us/product/image-lab-software?ID=KRE6P5E8Z |
| Prism 8 | Graph Pad | www.graphpad.com |
| CCTop tool | [82] | https://crispr.cos.uni-heidelberg.de |
| NIS-Elements Advanced Research software | Nikon | N/A |
| FlowJo software v9.7 | Becton Dickinson | https://www.flowjo.com |
| Other | ||
| LPS from Salmonella enterica | Sigma-Aldrich | L6143 |
| HDM extract (Dermatophagoisdes farina) | Greer Laboratories Inc./Fisher Scientific | NC0277827 |
AB, antibody; ACK, ammonium-chloride-potassium; BSA, bovine serum albumin; CSL, CBF1/Suppressor of Hairless/LAG-1; DSS, decxtran sodium sulfate; EMSA, electrophoretic mobility shift assay; FBS, fetal bovine serum; HDM, house dust mite; HRP, horseradish peroxidase; IgG, immunoglobulin G; IgM, immunoglobulin M; LPS, lipopolysaccharide; mAb, monoclonal antibody; N1, Notch1; N1ΔE, constitutive active Notch1 lacking extracellular domain; N2, Notch2; qPCR, quantitative PCR; RA, Arg-Ala substitution; RPMI, Roswell Park Memorial Institute; sgRNA, short guide RNA; SPS, sequence-paired site; TMB, 3,3′,5,5′-Tetramethylbenzidine; UNLB, unlabeled.