Spinx column

240 μl of lysate was incubated with anti-Flag antibody coated magnetic beads and 10000 units of RNase T1 (ThermoFisher scientific, EN0541) to perform digestion of exposed mRNA and ribosome purification simultaneously. 100 μl of Dynabeads protein G coated with 4 μg anti-Flag antibody was used. The lysate-beads-RNase T1 mixture was incubated at 4°C for 6 hours and washed; RNA was extracted as described above.
To perform size selection, the extracted RNA sample was separated on a denaturing 15% polyacrylamide urea gel. The gel region corresponding to 30–45 nt, as estimated by oligo markers, was excised. The gel slice was homogenized in 500 μl elution buffer (10 mM Tris-HCl, PH 7.5, 250 mM NaCl, 1 mM EDTA) supplemented with 0.2% SDS and RNAsecure reagent (ThermoFisher scientific, AM7005). The gel slurry was heated at 60°C for 10 min to allow inactivation of contaminating RNase by RNAsecure reagent and transferred to 4°C for overnight elution of RNA from the gel. The eluate was collected by centrifuging the gel slurry through a Spin-X column (Sigma, CLS8162), and RNA was precipitated by adding an equal volume of isopropanol and 25 μg linear acrylamide, incubated at room temperature for 30 min, and centrifuged for 15 min at 17000Xg, 4°C. The pellet was air dried and dissolved in 15 μl RNase-free water.

In vitro transcribed HCV (1-117) RNAs were ethanol precipitated, resuspended in RNase-free H₂O and treated with DNase I (New England Biolabs) to remove the DNA template, followed by purification with Sephadex G-25 spin columns (Cytiva) to remove unincorporated NTPs and the degraded template. The flowthrough was treated with quickCIP (New England Biolabs) to remove the 5′ triphosphate followed by heat inactivation of the phosphatase (2 min at 80 °C). Viral RNAs were labeled with T4 PNK (New England Biolabs) and γ[³²P]ATP followed by gel purification on a denaturing (8 M urea) 15% acrylamide gel run in 0.5× Tris-borate-ethylenediaminetetraacetic acid (TBE) buffer. Bands were imaged, excised, and crushed in 300 µL crush and soak buffer (0.5 M NH₄OAc, 0.2% sodium dodecyl sulfate, and 1 mM ethylenediaminetetraacetic acid [EDTA]) followed by elution at 4 °C overnight. The gel was removed with a spin-X column (Sigma Aldrich), and the RNA was precipitated using 3M NaOAc, pH 5.2 and ice-cold 100% ethanol at −20 °C for >20 min. RNAs were pelleted by centrifugation, washed with 150 µL ice-cold 75% ethanol, dried, resuspended in RNase-free H₂O, and quantified by nanodrop spectrophotometer. Extinction coefficients were calculated with the IDT online tool available at https://www.idtdna.com.

GFP or Halo IP was performed according to the manufacturer’s protocol (for GFP IP: GFP-Trap Agarose—ChromoTek GmbH, for Halo IP: https://promega.co.uk/-/media/files/resources/protocols/technical-manuals/0/halolink-resin-protocol.pdf) as described in (77 (link)). Briefly, lysates were incubated with either GFP-Trap Agarose beads (Chromotek) or HaloLink Resin (Promega) for 1 to 2 hours (20 μl of packed resin/1 mg of lysate). Immunoprecipitates were washed three times with wash buffer [50 mM tris-HCl (pH 7.5) and 150 mM NaCl] and eluted by adding 2× NuPAGE LDS sample buffer. The mixture was then incubated at 95°C for 10 min, and the eluent was collected by centrifugation through a 0.22-μm Spin-X column (CLS8161, Sigma-Aldrich). Eluted samples were supplemented with 1% (by volume) β-mercaptoethanol and denatured at 70°C for 10 min before being subjected to immunoblot analysis.

Cells were transiently transfected as described above. Twenty-four hours post transfection, cells were harvested in lysis buffer and lysates clarified by centrifugation at 17,000g for 10 min at 4°C. An amount of 500 µg of whole-cell lysate was incubated with 15 µl of packed beads of either nanobody anti-GFP binder-Sepharose or anti-HA frankenbody-Sepharose beads (generated by the MRC PPU Reagents and Services) for 1 h. Bound complexes were recovered by washing the beads three times with 50 mM Tris–HCl pH 7.5 150 mM NaCl before eluting with 2× SDS–PAGE sample buffer supplemented with 0.1% (by vol) 2-mercaptoethanol. The samples were denatured at 70°C for 10 min and the resin was separated from the sample by centrifugation through a 0.22 µm Spin-X® column (CLS8161, Sigma).

Lipid-loaded CD1 tetramers were generated as published previously.¹⁹ (link) Briefly, glucose monomycolate (GMM) or synthetic diacylated sulfoglycolipid (Ac₂SGL) was sonicated into pH 4.50 mM sodium citrate buffer, containing 0.25% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). CD1b monomer was added to either a 20-fold (GMM) or 40-fold (Ac₂SGL) molar excess of lipid. Monomer-lipid suspensions were incubated overnight at 37 °C and neutralized to pH 7.4 with 1 M Tris pH 9. Lipid-loaded monomers were tetramerized by adding 1.25 molar equivalents of fluorophore-conjugated streptavidin (Supplementary Table S2) at a 4:1 ratio of monomer to streptavidin at room temperature over 2 h then filtered through a SpinX column (Sigma-Aldrich Cat#CLS8162). Mock-loaded CD1b tetramers were generated by the same procedure without exogenous lipid. CD1d-PBS-57 (α-galactosylceramide or α-GalCer) PE and MR1-5-OP-RU BV421 were used as provided by the National Institutes of Health Tetramer Core Facility (Emory University, Atlanta, GA). Tetramers were validated by staining with a positive control T cell line.

For co-immunoprecipitation the cells were lysed in lysis buffer and 1 mg of total protein was incubated with 7 μg ULK1 antibody for 60 min at 4°C under rotation. 50 μl slurry of Protein A/G Resin (Expedeon) were added, followed by incubation for 60 min at 4°C under rotation. Beads were then washed three times in PBS and once in lysis buffer and proteins were eluted from beads with 2× LDS, followed by heating at 95°C for 5 min. Eluates were passed through a Spin-X column (Sigma-Aldrich) before loading on an 8% Tris-glycine gel and Western blot analysis.

PBS-57 (α-galactosylceramide or α-GalCer)-loaded and unloaded human CD1D monomers were provided by the National Institutes of Health Tetramer Core Facility (Emory University, Atlanta, GA). Tetramers were prepared as previously described (Liu et al. 2006 (link)). Briefly, 10 μL of the loaded or unloaded stock monomers was incubated with 19 μL of streptavidin conjugated to APC (Life Technologies, Carlsbad, CA), PE (Life Technologies, Carlsbad, CA), DyLight 488 (Thermo Fisher Scientific, Rockford, IL), or BV650 (BioLegend, San Diego, CA) that were titrated in at ten aliquots of 1.9 μL every 10 min to facilitate tetramerization. The tetramer was filtered through a SpinX column (Sigma, St. Louis, MO) to remove aggregates and stored at 4 °C until use.