Restriction and modification enzymes

C1q was purified from human serum as described by Arlaud et al. (35 (link)). C1q-collagen-like regions (CLR) and globular regions (GR) were prepared as described previously (36 (link)). MW and A_{1%, 1cm} at 280 nm used for protein quantification were 459,300 g/mol and 6.8 for C1q, 189,900 g/mol and 2.1 for C1q-CLR, and 48,000 g/mol and 9.3 for C1q-GR. Recombinant human CRT was produced and quantified (MW = 49,431 g/mol and A_{1%, 1cm} = 16.5) as described by Païdassi et al. (28 (link)). Low density lipoprotein (LDL) was from Sigma-Aldrich and AcLDL was from Acris Antibodies. LDL and AcLDL concentrations were determined using the Quick Start Bradford 1x Dye Reagent (Biorad). The protein contents represent approximately a quarter of the total weight of the LDL samples. Alexa Fluor 568-succinimidyl ester was obtained from Molecular probes. Recombinant human CRT labeling with the succinimidyl ester conjugate was performed according to the manufacturer's protocol. N-glycosidase F (PNGase F) was purified from cultures of Flavobacteriummeningosepticum according to the method of Tarentino et al. (37 (link)), with the modification by Aude et al. (38 (link)).
Oligonucleotides were from Eurogentec. The list of primers used is provided in Table S1. Restriction and modification enzymes were from New England Biolabs. The pCMV6-SR-F1 plasmid was from Origene. Pfu polymerase was from Stratagene.

DNA manipulations were performed with standard techniques. Restriction and modification enzymes were purchased from New England Biolabs. Preparations of plasmid DNA were carried out with a NucleoBond Xtra Midi plasmid purification kit (Macherey-Nagel).

Chemicals were purchased from VWR, Lutterworth, Leicestershire, United Kingdom, or Sigma-Aldrich Co. Ltd., Poole, Dorset, United Kingdom, and were the purest grades available. Restriction and modification enzymes were purchased from New England BioLabs, and DNA purification reagents were obtained from Qiagen.

Plasmid pET19mod_rfaH-F51C-S139C encoding RfaH^CC was generated by successive site-directed mutagenesis according to the QuickChange Site-Directed Mutagenesis Kit protocol, using pET19mod_rfaH as a template^{33 (link)}. All other plasmids were constructed by standard molecular biology approaches with restriction and modification enzymes from New England Biolabs. Sequences of all plasmids were confirmed by Sanger sequencing either at the Genomics Shared Resource Facility, Ohio State University, USA or at Eurofins Genomics, Germany.

C1q was purified from human serum and quantified as described (12 (link)). Human serum was obtained from the Etablissement Français du Sang (EFS) Rhône-Alpes (agreement number 14-1940 regarding its use in research). C1q collagen stalks (CLR) and C1q globular heads (GR) were prepared according to Tacnet-Delorme et al. (13 (link)). The recombinant protease tetramer C1r2s2 was produced and purified according to Bally et al. (14 (link)). Full-length LRP1 (soluble LRP1) was produced and purified according to De Nardis et al. (15 (link)). Recombinant C1q and C1q mutant LysA59Ala/LysB61Ala/LysC58Ala were produced and purified as described in Bally et al. (16 (link)). For protein quantification, Mw and A_{1%, 1 cm} were respectively for C1q (459,300; 6.8) (12 (link)), CLR (189,900; 2.1), GR (48,000; 9.3) (17 (link)), C1r2s2 (330,000; 13.5) (18 (link)). Oligonucleotides were from Eurogentec. Restriction and modification enzymes were from New England Biolabs.
Full-length LRP1 Myc DDK clone (RC218369) was purchased from Origene. The pET22B-RAP plasmid was kindly provided by Søren Moestrup, Aahrus University, Denmark. pcDNA3.1 mini IV HA-tag was cloned as previously indicated (19 (link)).

Plasmids used in this study are shown in Figure S1. The SARS-CoV-2 nsp7/8/12^A genes were codon-optimized for expression in E. coli and synthesized by GenScript and subcloned into standard pET-derived expression vectors under control of the T7 gene 10 promoter and lac repressor. The derivative plasmids were constructed by standard molecular biology approaches with restriction and modification enzymes from New England Biolabs, taking advantage of the existing or silent restriction sites engineered into the Nsp12 coding sequence. DNA oligonucleotides for vector construction and sequencing were obtained from Millipore Sigma. Sequence of all plasmids, including pET22a-Nsp12, were confirmed by Sanger sequencing at the Genomics Shared Resource Facility (the Ohio State University), and will be available upon request.