Peg ittm virus precipitation solution

Luciferase genes (Luc) from pGL4.10[luc2] or pGL4.12[luc2CP] (Promega, Madison, USA) were replaced with the Luc sensor region of the pWPXL: 5×GRE-Luc lentiviral transfer vector (Lee et al., 2016 (link)). The elongation factor 1α (EF1α) promoter was replaced by the 5×GRE sequences of the 5×GRE-Luc2CP lentiviral transfer vectors to serve as a control reporter constitutively expressing luciferase. The lentivirus was produced by co-transfecting the transfer vector, second-generation packaging vector (psPAX2, Addgene plasmid 12260), and an envelope plasmid (pMD2.G, Addgene plasmid 12259) at a ratio of 2:1:1 into HEK-293T cells [American Type Culture Collection (ATCC), Manassas, USA] using the Effectene reagent (Qiagen). Viral supernatants were collected 48 h after transfection, passed through a 0.2-μm filter, and concentrated by centrifugation using PEG-itTM virus precipitation solution (System Biosciences, Palo Alto, USA). Aliquots were stored in phosphate-buffered saline (PBS) in the presence of TransDux^TM MAX Lentivirus Transduction Enhancer (System Biosciences) and stored at −80°C until use. The titers of lentivirus were in the range of 1.50–1.95×10¹² genome copies/ml.

Total cellular mRNAs expressed in human T cell line persistently infected with HIV-1IIIB strain, Molt-4/IIIB cells23 (link) were isolated by using Isogen-LS reagent (Isogen, Nippon Gene, Toyama, Japan) followed by oligo-(dT) column purification (Oligotex™-dT30, Takara & Clontech Laboratories Inc. Japan). For preparation of HIV-1 genomic RNAs in virus particles, culture supernatant of Molt-4/IIIB cells was harvested and filtered through 0.45-μ-pore-size filters. Then, virus particles in the culture supernatant were precipitated by using PEG-it system according to the manufacture’s protocol (PEG-itTM virus precipitation solution, System Biosciences, CA, USA). Total mRNA from the virus particles was similarly isolated. To remove the cap moiety of mRNA, each isolated mRNA fraction was treated with Tobacco Acid Pyrophosphatase (TAP, Nippon Gene, Toyama, Japan) in the reaction buffer containing 200 units of TAP, 50 mM sodium acetate, 5 mM EDTA and 10 mM 2-mercaptoethanol for 37 °C for 60 min.

Gene knockout in human colonoid was achieved using the CRISPR-Cas9 system, following a published protocol^{42 (link)} with some modifications. Briefly, MYADM-sgRNA was cloned into lentiCRISPRv2 (Addgene, #52961), and the plasmid was used for lentivirus production. Lentivirus solution was collected and concentrated using PEG-it^TM Virus Precipitation Solution (System Biosciences) following the manual. The virus pellet was then resuspended in 600 µL CMGF(-) medium that consists of Advanced DMEM/F12 medium supplemented with 100 U/mL penicillin-streptomycin, 10 mM HEPES buffer and 1× Glutamax and stored at −80 °C.