KEY RESOURCES TABLE
| REAGENT or RESOURCE | SOURCE | IDENTIFIER |
|---|---|---|
| Antibodies | ||
| β-Actin (8H10D10) Mouse mAb (1:9000 for WB) | Cell Signaling Technology | 3700S RRID: AB_2242334 |
| Anti-beta Actin antibody (1:1000 for WB) | Abcam | ab8227 RRID: AB_230518: |
| Monoclonal ANTI-FLAG® M2 antibody produced in mouse (1:1000 for WB) | Sigma-Aldrich | F1804 RRID: AB_262044 |
| Anti-YTHDF1 antibody (1:1000 for WB) | Abcam | ab157542 RRID: N/A |
| YTHDF2 Polyclonal Antibody (1:250 for WB) | Thermo Fisher Scientific | PA5–63756 RRID: AB_2649742 |
| Recombinant Anti-METTL3 antibody [EPR18810] (1:1500 for WB) | Abcam | ab195352 RRID: AB_2721254 |
| Rig-I (D14G6) Rabbit mAb (1:200 for IF) | Cell Signaling Technology | 3743S RRID: AB_2269233 |
| Ub-K63 Monoclonal Antibody (HWA4C4) (1:200 for IF) | Thermo Fisher Scientific | 14–6077-82 RRID: AB_1257213 |
| Mouse Anti-YTHDF2 polyclonal antibody (1:200 for IF) | USBiological Life Sciences | 135486 RRID: N/A |
| IRDye® 800CW Goat anti-Mouse IgG Secondary Antibody (1:15,000 for WB) | Li-COR Biosciences | 926–32210 RRID: AB_621842 |
| IRDye® 800CW Goat anti-Rabbit IgG Secondary Antibody (1:15,000 for WB) | Li-COR Biosciences | 926–32211 RRID: AB_621843 |
| IRDye® 680RD Goat anti-Mouse IgG Secondary Antibody (1:15,000 for WB) | Li-COR Biosciences | 926–68070 RRID: AB_10956588 |
| IRDye® 680RD Goat anti-Rabbit IgG Secondary Antibody (1:15,000 for WB) | Li-COR Biosciences | 926–68071 RRID: AB_10956166 |
| Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 594 (1:2000 for IF) | Thermo Fisher Scientific | A32740 RRID: AB_2762824 |
| Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 647 (1:2000 for IF) | Thermo Fisher Scientific | A32728 RRID: AB_2633277 |
| m6A Polyclonal rabbit purified antibody | Synaptic Systems | 202 003 RRID: AB_2279214 |
| Anti-FLAG® M2 Magnetic Beads | Sigma-Aldrich | M8823 RRID: AB_2637089 |
| Brilliant Violet 711™ anti-mouse CD8a Antibody (clone 53–6.7) | Biolegend | 100747 RRID: AB_11219594 |
| Brilliant Violet 785™ anti-mouse CD3 Antibody (clone 17A2) | Biolegend | 100231 RRID: AB_11218805 |
| Brilliant Violet 650™ anti-mouse CD4 Antibody (clone RM4–5) | Biolegend | 100555 RRID: AB_2562529 |
| Purified Rat Anti-Mouse IFN-γ (clone XMG1.2) | BD Biosciences | 554409 RRID: AB_398550 |
| Brilliant Violet 421™ anti-mouse CD11c Antibody (clone N418) | Biolegend | 117329 RRID: AB_10897814 |
| Brilliant Violet 650™ anti-mouse/human CD11b Antibody (clone M1/70) | Biolegend | 101239 RRID: AB_11125575 |
| Alexa Fluor® 700 anti-mouse I-A/I-E Antibody (clone M5/114.15.2) | Biolegend | 107621 RRID: AB_493726 |
| FITC anti-mouse CD86 Antibody (clone PO3) | Biolegend | 105109 RRID: AB_313162 |
| Brilliant Violet 605™ anti-mouse CD45 Antibody (clone 30-F11) | Biolegend | 103155 RRID: AB_2650656 |
| Goat Anti-Mouse IgG(H+L)-HRP | Southern Biotech | 1036–05 RRID: AB_2794348 |
| Goat Anti-Mouse IgG1-HRP | Southern Biotech | 1071–05 RRID: AB_2794426 |
| Goat Anti-Mouse IgG2c-HRP | Southern Biotech | 1078–05 RRID: AB_2794462 |
| Bacterial and Virus Strains | ||
| NEB® Turbo Competent E. coli (High Efficiency) | New England Biolabs | C2984H |
| Biological Samples | ||
| Chemicals, Peptides, and Recombinant Proteins | ||
| In-Fusion® HD Cloning Kit | Takara Bio | 638909 |
| Fluorescein-12-UTP | Sigma-Aldrich | 11427857910 |
| N6-Methyladenosine-5’-Triphosphate | TriLink Biotechnologies | N-1013 |
| Pseudouridine-5’-Triphosphate | TriLink Biotechnologies | N-1019 |
| Inosine-5’-Triphosphate | TriLink Biotechnologies | N-1020 |
| FastAP | Thermo Fischer Scientific | EF0652 |
| RNaseR | Lucigen | RNR07250 |
| RNA Loading Dye, (2X) | New England Biolabs | B0363S |
| GeneRuler 1 kb DNA Ladder, ready-to-use | Thermo Fischer Scientific | SM0313 |
| RiboRuler Low Range RNA Ladder, ready-to-use | Thermo Fischer Scientific | SM1833 |
| T4 DNA Ligase | New England Biolabs | M0202M |
| DNase I | Ambion | AM2222 |
| T4 PNK | New England Biolabs | M0201S |
| RNA Fragmentation Reagents | Thermo Fischer Scientific | AM8740 |
| Ribolock RNase Inhibitor | Thermo Fischer Scientific | EO0382 |
| TRIzol | Thermo Fischer Scientific | 15596018 |
| Phenol:Chloroform:Isoamyl Alcohol 25:24:1 Saturated with 10 mM Tris, pH 8.0, 1 mM EDTA | Sigma-Aldrich | P2069 |
| cOmplete™ Protease Inhibitor Cocktail | Sigma-Aldrich | 11697498001 |
| Dynabeads™ Protein A for Immunoprecipitation | Thermo Fischer Scientific | 10002D |
| PEG 8000, Molecular Biology Grade (Polyethylene Glycol 8000) | Promega | V3011 |
| DAPI | Thermo Fischer Scientific | D1306 |
| Annexin V Binding Buffer, 10X concentrate | BD Biosciences | 556454 |
| Annexin V, Alexa Fluor™ 647 conjugate | Thermo Fischer Scientific | A23204 |
| Falcon® 5mL Round Bottom Polystyrene Test Tube, with Cell Strainer Snap Cap | Corning | 352235 |
| Falcon® 40 µm Cell Strainer, Blue, Sterile, | Corning | 352340 |
| Polyinosine-polycytidylic acid -TLR3 agonist | InvivoGen | vac-pic |
| EndoFit Ovalbumin | InvivoGen | vac-pova |
| Two OVA peptide standards for ELISPOT | InvivoGen | vac-sin |
| in vivo-jetPEI | Polyplus transfection | 201–10G |
| Corning® Matrigel® Growth Factor Reduced (GFR) Basement Membrane Matrix | Corning | 356231 |
| D-Luciferin Firefly, potassium salt | Biosynth International | L-8220 |
| Histopaque®–1083 | Sigma-Aldrich | 10831 |
| BD GolgiPlug™ Protein Transport Inhibitor (Containing Brefeldin A) | BD Biosciences | 555029 |
| Nunc MaxiSorp™ flat-bottom | Thermo Fischer Scientific | 44–2404-21 |
| Bovine Serum Albumin | Sigma-Aldrich | A9418–5G |
| 1-Step™ Ultra TMB-ELISA Substrate Solution | Thermo Fischer Scientific | 34028 |
| Stop Solution for TMB Substrates | Thermo Fischer Scientific | N600 |
| Antibody Diluent | Thermo Fischer Scientific | 3118 |
| VECTASHIELD® Antifade Mounting Medium with DAPI | Vector Laboratories | H-1200 |
| Square Cover Glasses No. 1 1/2, Corning® | VWR | 89239–698 |
| Lipofectamine 3000 | Thermo Fischer Scientific | L3000008 |
| Opti-MEM™ I Reduced Serum Medium | Thermo Fischer Scientific | 31985088 |
| DMEM, high glucose, pyruvate | Thermo Fischer Scientific | 11995–073 |
| Trypsin-EDTA (0.25%), phenol red | Thermo Fischer Scientific | 25200056 |
| Penicillin-Streptomycin | Thermo Fischer Scientific | 15140–163 |
| HyClone Characterized Fetal Bovine Serum (FBS), U.S. Origin | Thermo Fisher Scientific | SH30071.03 |
| NuPAGE™ 4–12% Bis-Tris Protein Gels, 1.0 mm, 15-well | Thermo Fisher Scientific | NP0323BOX |
| NuPAGE™ MOPS SDS Running Buffer (20X) | Thermo Fisher Scientific | NP0001 |
| 2x Laemmli Sample Buffer | Bio-Rad Laboratories | 161–0737 |
| Phosphate Buffered Saline with Tween® 20 (PBST-20X) | Cell Signaling Technologies | 9809 |
| milliTUBE 1ml AFA Fiber | Covaris | 520130 |
| RIG-I purified protein | Peisley et al., 2013 | N/A |
| K63-Ubn purified protein | Dong et al., 2011 | N/A |
| MAVS CARD-S purified protein | Wu et al., 2016 | N/A |
| BIOMOL® Green | Enzo Life Sciences | BML-AK111–0250 |
| SNAP-Surface® Alexa Fluor® 647 | New England Biolabs | S9136S |
| 35S-IRF3 | Ahmad et al., 2018 | N/A |
| SYBR™ Gold Nucleic Acid Gel Stain (10,000X Concentrate in DMSO) | Thermo Fisher Scientific | S11494 |
| Critical Commercial Assays | ||
| ZymoPURE II Plasmid Prep Kits | Zymo Research | D4200 |
| MEGAscript™ T7 Transcription Kit | Thermo Fisher Scientific | AM1334 |
| Direct-zol RNA Miniprep | Zymo Research | R2051 |
| RNeasy Mini Kit | Qiagen | 74106 |
| RNA Clean & Concentrator-5 | Zymo Research | R1013 |
| Zymoclean Gel Recovery Kit | Zymo Research | R1011 |
| RiboMinus Eukaryote System v2 kit | Thermo Fisher Scientific | A15026 |
| Poly(A)Purist™ MAG Kit | Thermo Fisher Scientific | AM1922 |
| Brilliant II QRT-PCR Master Mix Kit, 1-Step | Agilent | 60080 |
| Neon™ Transfection System 100 µL Kit | Thermo Fisher Scientific | MPK10025 |
| RNA ScreenTape | Agilent | 5067–5576 |
| RNA ScreenTape Sample Buffer | Agilent | 5067–5577 |
| LIVE/DEAD™ Fixable Green Dead Cell Stain Kit, for 488 nm excitation | Thermo Fisher Scientific | L23101 |
| BD Cytofix/Cytoperm Fixation/Permeabilization Solution Kit | BD Biosciences | 554714 |
| Deposited Data | ||
| m6A-irCLIP sequencing data | This paper | GEO: GSE116007 |
| ChIRP-MS data | Chen et al., 2017 | N/A |
| Experimental Models: Cell Lines | ||
| HeLa | ATCC | CCL-2 |
| HEK293T | ATCC | CRL-3216 |
| RIG-I KO HeLa | Chen et al., 2017 | N/A |
| YTHDF2 KO HeLa | Dr. Chuan He | N/A |
| Experimental Models: Organisms/Strains | ||
| C57BL/6J mice | Jackson Laboratories | 664 |
| Oligonucleotides | ||
| qRT-PCR primers | This paper | Table S1 |
| irCLIP sequencing primers | Zarnegar et al., 2016 | N/A |
| SMARTpool ON-TARGETplus METTL3 siRNA | Dharmacon | L-005170–02-0005 |
| ON-TARGETplus Non-targeting Control siRNA #1 | Dharmacon | D-001810–01-05 |
| Recombinant DNA | ||
| Plasmid: autocatalytic-splicing linear GFP-IRES (circGFPd2IRES) | Chen et al., 2017 | N/A |
| Plasmid: circGFPd2IRES_N’5BoxB | This paper | pRC0050 |
| Plasmid: pPB-CAG-Flag-GGS-lambda | Dr. Chuan He | N/A |
| Plasmid: pPB-CAG-Flag-YTHDF1N | Dr. Chuan He | N/A |
| Plasmid: pPB-CAG-Flag-YTHDF1N-lambda | Dr. Chuan He | N/A |
| Plasmid: pPB-CAG-Flag-YTHDF2N | Dr. Chuan He | N/A |
| Plasmid: pPB-CAG-Flag-YTHDF2N-lambda | Dr. Chuan He | N/A |
| Plasmid: pCAGGS-Flag-YTHDF1N | This paper | pRC0070 |
| Plasmid: pCAGGS-Flag-YTHDF1N-GS-lambda | This paper | pRC0071 |
| Plasmid: pCAGGS-Flag-YTHDF2N | This paper | pRC0103 |
| Plasmid: pCAGGS-Flag-YTHDF2N-GS-lambda | This paper | pRC0104 |
| Plasmid: pCAGGS-3xFlag-YTHdomain | This paper | pRC0151 |
| Plasmid: pCAGGS-3xFlag-mRuby3-YTHdomain | This paper | pRC0152 |
| Plasmid: pCAGGS-3xFlag-YTHdomain-GS-lambdaN | This paper | pRC0164 |
| Plasmid: pCAGGS-3xFlag-mRuby3-YTHdomain-GS-lambdaN | This paper | pRC0165 |
| Software and Algorithms | ||
| ZEN (blue edition) | Carl Zeiss Microscopy | zeiss.com/microscopy/us/products/microscope-software/zen.html |
| FlowJo_V10 | FlowJo, LLC | flowjo.com/solutions/flowjo/downloads |
| Other | ||