Optima xpn 100 ultracentrifuge

The full-length TIALD sequence was synthesized by General Biol (China) and cloned into a lentiviral vector to construct a TIALD overexpression vector, pCDH-TIALD-puro-copGFP. To knock down the target genes, the same cDNA oligonucleotides with the shRNAs against TIALD, METTL16, YTHDF2, YTHDC1 were synthesized by Sangon Biotech (China) respectively. The sequences were listed in Supplementary Table S2. After annealed, double-strand oligonucleotides were inserted into pLKO.1 vector (Addgene). For transient transfection, cells were transfected using Lipofectamine 3000 (Thermo, USA) and harvested for the following assays after 48 h of transfection.
To construct stably modified cell lines, plasmids pCDH-TIALD-puro-copGFP and pLKO.1-shTIALD-EGFP were firstly applied to generated lentiviruses (LV-TIALD-GFP and LV-shTIALD-GFP) as previously described respectively [46 (link)]. Afterwards, lentiviral particles were concentrated by Optima XPN-100 Ultracentrifuge (Beckman Coulter, USA) at 15000 g for 2 h under vacuum at 4 °C, and then the concentrated lentiviral particles were resuspended by 500 μL PBS. SNU-449 and SMMC-7721 cells with 50% cell density in 6 well cell culture plates were infected by 100 μL lentivirus for 2 days in the presence of polybrene (1 μg/mL, Santa Cruz Biotechnology, USA). The infected cell lines were further selected by puromycin (2 μg/ml) and verified by qRT-PCR.

Exosomes were purified through standard differential centrifugation protocols as previously described [28 ]. In brief, exosomes were purified from the cultured medium at 4 °C by sequential centrifugation steps at 300 g (10 min), 2000 g (20 min), and 10,000 g (30 min) to eliminate cellular debris. The supernatant was filtered through a 0.22 mm filter to remove larger microvesicles (> 220 nm) and then further centrifuged (Optima XPN-100 Ultracentrifuge, Beckman, USA) at 100,000 g (90 min) to to extract exosomes. The exosome pellet was resuspended in PBS, centrifuged at 100,000 g at 4 °C for 90 min, and resuspended in 100 μl of PBS for further analysis. Importantly, the same concentrations of exosomes from the Ctrl-Exo and LINC00511-Exo groups were used in the indicated experiments.

The open reading frame of SFTSV M segment, tagged with a C-terminal Flag-peptide (DYKDDDDK), was synthesized by GenScript Biotech (China) and subcloned into pcDNA3.1(+) vector. The N914Q mutant was constructed using the Q5® Site-Directed Mutagenesis Kit (New England Biolab, USA). The WT and mutant pseudoviruses were prepared as previously described^{62 (link)}. Briefly, HEK293T/17 cells were co-transfected with pLentiCMV-Luc2, pCMV-dR8.2-dvpr and the WT/mutant M expressing plasmids using PEIpro transfection reagent (Polyplus Transfection, France). The cell culture supernatant containing virions was collected at 48 h post transfection, which was clarified by low-speed centrifugation (1000 × g, 10 min) and filtered with a 0.45-μm cut-off syringe filter to remove the cell debris. The cleared supernatant was then layered onto a 20% (wt/vol) sucrose cushion and purified by ultracentrifugation (SW41 rotor, 209,900 × g, 2 h, 4 °C) using an Optima XPN-100 ultracentrifuge (Beckman Coulter). The resulting pellet was resuspended in sterilized 1×PBS (pH 7.4) and was used for subsequent Western blotting and infection assays. To quantify the viral titer, equal volumes of WT/mutant pseudovirus solutions were used to infect HEK293 cells. At 48 hpi, luciferase activities were determined using the Bright-Lumi™ Firefly Luciferase Assay Kit (Beyotime, China).

The conditioned media was centrifuged at 300 × g for 10 minutes at 4°C to remove cell debris. Sequential centrifugation steps were performed to separate three EV populations as follows: 2,000 × g for 20 minutes at 4°C to collect apoptotic bodies (ABs), 16,500 × g for 30 minutes at 4°C to pellet microvesicles (MVs), and 100,000 × g for 90 minutes at 4°C for isolation of exosomes (EXs) (Optima XPN-100 Ultracentrifuge, Beckman Coulter, California, USA). EV pellets were resuspended in DMEM/F12 or PBS and stored at − 80°C for further usage.

HEK293T cells (ATCC, CRL-11268) were cultured in 10-cm tissue culture dishes until they reached 70–80% confluence, after which plasmids that contained the gene of interest (10 μg), psPAX2, and pMD2G (5 μg and 2.5 μg, respectively), were transfected at a ratio of 4:2:1 using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). After 8 h of transfection, the culture medium was exchanged with 12 ml of growth medium. The viral supernatants were collected 48 h after the initial transfection and filtered through a Millex-HV 0.45 μm low-protein-binding membrane (Millipore, Darmstadt, Germany) before being concentrated 1000-fold by ultracentrifugation (Optima XPN-100 Ultracentrifuge, Beckman Coulter) at 4 °C at 25,000 rpm for 2 h. The pellets were resuspended in phosphate-buffered saline (PBS) or growth medium. The titer of lentivirus was measured with Lenti-X p24 Rapid Titer Kit (Clontech, Mountain View, CA) according to the manufacturer’s instructions. The estimated infectious titers pf Cas9-only, Cas9-sgRNA, and dCas9-sgRNA viruses were 2.34 × 10¹⁰, 2.24 × 10¹⁰, and 2.20 × 10¹⁰ TU/ml, respectively. In addition, lentiviral vector constructs encoding Cas9-only (1.16 × 10⁹ TU/ml), Cas9-sgRNA (1.28 × 10⁹ TU/ml), and Cas9-sgRNA (1.02 × 10⁹ TU/ml) with CMV promoter were from VectorBuilder (Santa Clara, CA, USA).