Snabi

The sense and antisense RNAi fragments (FARn-S) from Ab-far-n (n: 2, 3, 5, 6, 7, 8) open reading frames (ORFs) were amplified using primers FARncm-F1/FARncm-R1 and FARncm-F2/FARncm-R2, respectively (Table S2). The sense PCR fragments were inserted into the vector pBS-1 digested with either XhoI or SnaBI (Thermo Fisher Scientific, Waltham, MA, USA), respectively. After sequencing, the corresponding antisense PCR fragments were inserted into the BglII and StuI (Thermo Fisher Scientific) sites to form the RNAi vector, pBS2-Ab-far-n, with the Ab-far-n hairpin structure (Figure 1A). The DNA ligases and systems used for vector construction were based on the In-Fusion^® HD Cloning Kit (Takara, Shiga, Japan). In the vector construction process, the PCR fragments and new vectors were sequenced to ensure the accuracy of the insertion sequences.

ICAM‐1‐GFP was described earlier (Kroon et al, 2018 (link)) and cloned into a lentiviral pLV backbone using SnaBI (ThermoFisher, FD0404) and XbaI (ThermoFisher, FD0684)/NheI (ThermoFisher, FD0973). To generate ICAM‐1 truncated sequences, Gibson cloning (NEB) was performed on the pLV‐ICAM‐1‐GFP plasmid. All constructs contain GFP as FP and the ICAM‐1 signal peptide, consisting of amino acids Met1 to Ala27. ICAM‐1 Δ 1 is truncated from Gln28 to Val109; ICAM‐1 Δ 12 has a deletion from Gln28 to Phe212; ICAM‐1 Δ 123 lacks Gln28 to Ile307; ICAM‐1 Δ3 is truncated from Val213 until Ile307; ICAM‐1 Δ 4 lacks Pro311 to Arg391; ICAM‐1 Δ C terminates at Asn504. pLV‐ICAM‐2‐mKate was constructed and packaged by VectorBuilder (Vector ID is VB200624‐1164vtm). pLV‐mNeonGreen‐Caax and pLV‐mScarletI‐CAAX have been described earlier by us (Arts et al, 2021 (link)). mEos4b‐N1 was a gift from Micheal Davidson (Addgene # 54814; http://n2t.net/addgene:54814; RRID:Addgene_54814; Paez‐Segala et al, 2015 (link)). mEos4b was PCRed out of the mEos4b‐N1 vector, after which it was ligated into a pLV backbone using SnaBI and XbaI/NheI. All primers used in cloning are shown in Appendix Table S1.

Aliquots of phage suspension (10¹¹–10¹² PFU/ml) were subjected to phenol/chloroform extraction and ethanol precipitation as described by Carlson and Miller [38] . Isolated phage DNA was subsequently used in restriction analysis, for PCR or was subjected to genome sequencing. Restriction digestion was performed with BamHI, EcoRI, EcoRII, EcoRV, HindIII, KpnI, MboI, NheI, NotI, PstI, PvuI, SnaBI, SspI, VspI and XbaI restriction endonucleases (Fermentas) according to the supplier's recommendations. DNA fragments were separated by electrophoresis in a 0.8% agarose gel containing ethidium bromide. Restriction analysis was performed in triplicate to confirm the results.