Pirespuro3 vector

We designed two expression constructs encoding the SARS-CoV2 Receptor Binding Domain (RBD) (isolate Wuhan Hu-1) modified with a flexible linker, a SpyTag motif ^{17 (link)} and a 6xHis-tag on the N- or C-terminus, named His-Spy-RBD and RBD-Spy-His, respectively (Figure 1A). Both constructs had a SARS-CoV2 signal peptide (SP) on their N-terminus, and were flanked by a 5’ EcoRI and 3’ BamHI site for cloning. Both constructs were synthesized by IDT (Coralville, IA, USA) and cloned into the pIRESpuro3 vector (cat# 631619, Takara Bio, USA) using EcoRI and BamHI restriction enzymes (R0101S and R0136S respectively, New England Biolabs) and a Rapid Ligation Kit (cat# K1423, Thermo Fisher, USA). The constructs were validated by Sanger sequencing using CMV-F primers (GENEWIZ, South Plainfield, NJ, USA). For expected amino acid sequences, see Supplemental Information 1.

Horse genomic DNA was obtained from Zyagen (San Diego, USA). Equine chemokine receptors CXCRA, CXCR2, CCR2, CCR5, C5aR1, and the predicted Duffy antigen receptor (DARC) were amplified from equine genomic DNA by PCR using PfuTurbo DNA polymerase (Stratagene). Primers and accession numbers are listed in Supplementary Table 6. Exons encoding DARC were assembled using overlap extension PCR. All coding sequences were cloned into the pIRESpuro3 vector (Clontech) according to methods described elsewhere12 (link). The human Ga16 cDNA (pCISG16 plasmid) was kindly provided by Melvin I. Simon47 (link). The Ga16 gene was recloned in between the BstBI and EcoRV sites of the pIREShyg3 vector (Clontech) using the following primers:
5′-AACTATTTCGAAGCCGCCACCATGGCCCGCTCGCTGACCTG-3′ and
5′-ATCGAGGATATCTCACAGCAGGTTGATCTCGTC-3′.

As previously mentioned in the methods section 2.6, a 19 bp region of the Ads open reading frame (5′- ^{nt. 547}AACAAATATGAGCGTCTGA^{nt. 565}-3′) was targeted by the Ads specific shRNA. Using site-directed mutagenesis a new, mutated, Ads open reading frame was constructed where the same 19 bp region reads as follows: 5′- ^{nt. 547}AACAAGTACGAACGGCTCA^{nt. 565}-3′. These nucleotide substitutions (A⁵⁵²-G, T⁵⁵⁵-C, G⁵⁵⁸-A, T⁵⁶¹-G, G⁵⁶⁴-C) lead to five silent mutations such that the final amino acid sequence of both the original and mutated sequences was the same (N-K-Y-E-R-L). The newly constructed mut-Ads was cloned into the BamHI and BstBI restrictions site of pIRESpuro3 vector (Clontech, Mountain View, CA). Two million Ads KD Clone F cells were transiently transfected with 2 µg of pIRESpuro3-mut-Ads using Amaxa nucleatransfector and solution V (Program D-032). One fraction was stimulated for 4 days with sRANKL (60 ng/ml) and the cells were harvested for Western blot analysis. The other fraction was stimulated for 5 days with sRANKL (60 ng/ml) and the cells were processed for TRAcP staining.

Full-length human tau (0N4R) was cloned into the pcDNA3.1 vector acquired from human neuroblastoma cell line SH-SY5Y cDNA. A site-directed mutation at P301L was introduced in the construct using the QuikChange Lightning Multi Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA, USA). The sequence of human tau with the P301L mutation was then subcloned into the pIRESpuro3 vector (Clontech Laboratories, Mountain View, CA, USA).
HEK293T cells were cultured in Dulbecco’s modified Eagle’s medium (Nacalai Tesque) supplemented with 10% (v/v) fetal bovine serum (Hyclone, Logan UT, USA), and kept at 37 °C under a humidified atmosphere of 5% CO₂/95% air. Cells were seeded at 1 × 10⁵ cells/well in a 24-well plate. At 24 h after plating, the cells were transfected with the pIRESpuro3 vector containing human tau with the P301L mutation using Lipofectamine 3000 reagent (Thermo Fisher Scientific). After a 24-h incubation period, the cells were transferred to a 6-cm culture dish, and stable cell lines were selected with 1 µg/mL puromycin (Nacalai Tesque) for 12 days. Single cells from resistant colonies were transferred into the wells of 96-well plates.

Human DNMT isoforms 3B1, Δ3B2, 3B3, Δ3B4 and 3L, containing a MYC-tagged DNA sequence ligated to the 5′- ends, were amplified from pIRESpuro/Myc constructs48 (link) (a modified version of the pIRESpuro3 vector, Clontech), a gift from Dr Allen Yang (USC). Catalytically inactive mutants containing a cysteine to serine alteration in position 651 of DNMT3B1 and 452 of DNMTΔ3B2 proteins were established as previously described35 (link). MYC-tagged DNMT sequences were cloned into pLJM1 lentivirus vector at EcoRI and AgeI sites using Infusion HD PCR Cloning Plus (Clontech) following the manufacturer's protocol. To produce lentivirus for the specific constructs, the vesicular stomatitis virus envelope protein G-expression construct pMD.G1, the packaging vector pCMV ΔR8.91 and transfer vector pLJM1 were used as previously described49 (link). All vectors were amplified and purified using the PureYield Plasmid Maxiprep system (Promega), according to the manufacturer's instructions. The HCT116 derivative cell lines 3BKO, 3ABDKO and DKO8 were stably transfected with a lentivirus and selected with 2 μg ml⁻¹ puromycin for 14, 14 and 21 days, respectively.