Slhv033rs

shRNAs against Brd2, Brd3, Brd4, and Cbp were designed as described (52 (link), 53 (link)). Individual shRNAs were subcloned into the Hpa I/Xho I sites of the pLLX lentiviral vector. To generate lentivirus, lentiviral constructs containing the indicated shRNAs in the pLLX-shRNA-GFP, along with the helper plasmids Δ8.9 and vesicular stomatitis virus (VSV-G), were cotransfected into human embryonic kidney (HEK) 293T cells using polyethylenimine (PEI; 1 mg/ml; Polysciences, Warrington, PA; 23966). NB supplemented with B-27 and GlutaMAX was completely changed on the next day. Lentivirus transduction was carried out for additional 48 to 72 hours. Lentivirus containing media was filtered by a syringe filter (0.45 μm; Millipore, SLHV033RS) and directly used to infect neurons at DIV 3 and harvested at DIV 7. KD efficiency was measured by RT-qPCR and Western blotting. See table S6 for complete oligonucleotide list.

To optimize conditions for viral transduction, virus was produced in parallel in 10 cm dish- and 96-well format. For 10 cm dish-format, lentiviral particles were produced by transfecting HEK293T packaging cells at 80% confluence with 5 μg of the sgRNA lentiviral construct, 3.75 μg of psPax2 packaging plasmid, 1.25 μg of pMD2G envelope plasmid and 1 μg pAdVantage (Promega E1711) at a ratio of 3:1 DNA to FugeneHD (Promega E2311). For 96-well format, the number of cells and the amount of plasmid DNA was reduced 40 times. Approximately 24 h and 48 h after transfection, the supernatant containing viral particles was recovered, filtered through a 0.45 μm filter (Millipore SLHV033RS or MSHVS4510), pooled together, and frozen at -80^oC. In order to determine the multiplicity of infection (MOI) of the virus, 1 × 10⁵ fibroblasts were seeded per well of a 6-well plate and infected with serial dilutions of virus generated in 10 cm dish-format, starting from 1:1. Following selection with 4 μg/mL blasticidin, the number of viable cells/well were measured and compared to a non-infected control to calculate the percentage of infected cells. The formula P = 1-e^−m, where P is percentage of infected cells and m represents MOI, was used to determine the viral concentration yielding the desired MOI in 96-well format (typically MOI = 1).

Lenti-X 293T cells were co-transfected with pLKO.1 lentiviral plasmid, psPAX2, and pMD2.G using Lipofectamine LTX and PLUS Reagents (Thermo Fisher Scientific, 15338–100). After overnight incubation, the medium was changed to a fresh medium, and 60 to 72 h after transfection, the supernatant was collected and filtrated through a 0.45 μm filter (Millipore, SLHV033RS). The virus was concentrated using Lenti-X Concentrator (Clontech, 631231), resuspended by one-twentieth volume of PBS, and stored at −80°C before use. For infection of cultured neurons, one-hundredth volume of virus solution was added to the neuron culture medium, and 8 h after infection, the medium was changed to fresh medium. For mock infection, pLKO.1 empty vector was used as a lentiviral plasmid.

Reprogramming experiments followed the previously published protocol.²³ The four retroviral vectors (pMXs‐OCT4, SOX2, C‐MYC and KLF4) were introduced into plat‐E cells using lipofectamine 2000 transfection reagent (Thermo Fisher, 11668019) according to manufacturer's recommendations. After overnight transduction, the medium was replaced. Another 24 hours later, the virus‐containing supernatants were collected and filtered through a 0.45 mm cellulose acetate filter (Millipore, SLHV033RS), supplemented with 4 μg/ml polybrene (Millipore, TR‐1003‐G). MEF cells (seeded at 2 × 10⁵ cells per 10 cm culture dish) were incubated with virus‐containing supernatants for 48 hours with medium change at 24 hours before replacement with regular media. Two days after infection, the infected MEF cells were replaced with 2.5 × 10⁴ cells per 35 mm dish on mitomycin‐C‐treated MEF feeder layers.

Stocks of VSV-G pseudotyped HIV-1 pseudoparticles were prepared by transfecting 3 µg of pMD2.G, 12 µg of pSiCoR-luciferase, and 6 µg of pCMV-dR8.91 vectors into 293T cells using the standard JetPEI transfection protocol (PolyPlus, Illkirch-Graffenstaden, France). At 48 h post transfection, cell culture supernatants were collected, clarified on a 0.45 µm PVDF filter (Millipore SLHV033RS, Burlington, MA, USA), and concentrated twice by a Vivaspin20 centrifugal concentrator (Sartorius, VS2031, Göttingen, Germany). Supernatants containing pseudoparticles were stored in 500 µL aliquots at −80 °C. In parallel, viral stocks were titrated by anti-p24 enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s instructions (Innotest^® HIV Antigen mAb, Fujirebio, Tokyo, Japan).

Knockout sgRNAs were designed according to online software CHOPCHOP (https://chopchop.cbu.uib.no/) and cloned into LentiCRISPRv2 vector. lentiviral plasmid, psPAX2, and pMD2.G were transfected into HEK293T cells in 6-well plates by Turbofect transfection Reagent (R0532, Thermo Fisher Scientific). The supernatants were collected and filtered (SLHV033RS, Millipore) 48 h after transfection. Lentiviruses were used for melanoma cell infection. Melanoma cells with stable knockdown or overexpression were selected with 1 μg/ml puromycin. YAP1-V5 in pLX304 (Addgene #25890) was used for YAP1 overexpression. Duplexes of siRNA were synthesized by Genepharma (Shanghai, China). Transfection of siRNA was performed according to manufacturer's instructions. Non-targeting siRNA was used as a control.
The sequences of the sgRNAs were as follows:
BRD4: 1: 5'-AGACCAACCAACTGCAATACCT-3' and 2: 5'-GAGTCTGGGATGTTCGTCTCTC-3'; and YAP1: 1: 5'-GTGCACGATCTGATGCCCGG-3' and 2: 5'-ACATCGATCAGACAACAACA-3'.
The sequences of the siRNAs were as follows:
YAP1: 1: sense 5'-CUGCCACCAAGCUAGAUAATT-3', anti-sense 5'-UUAUCUAGCUUGGUGGCAGTT-3'; and YAP1: 2: sense 5'-GCAUCUUCGACAGUCUUCUTT-3', anti-sense 5'-AGAAGACUGUCGAAGAUGCTT-3'.