Plko 1 puro cmv turbogfp

At 24 hours prior to transfection, HEK293T cells were plated in 6-well plate at 8 × 10⁵ cells per well
in DMEM (10 % v/v FBS). Cells growing at ~70-80 % confluency were transfected with 1 μg of pLKO.1-puro-CMV-TurboGFP
(Sigma Aldrich) along with 2^nd generation lentiviral packaging and envelope plasmids, 0.75 μg of psPAX2 and 0.25
μg of pMD.2g (gifts from D. Trono, Addgene #12260, #12259). At 36 hours post transfection, the media containing lentiviral
particles was collected and passed through a 0.45 μm filter. Polybrene was added to a final concentration of 4
μg/mL. 3D hepatocyte colonies released from matrigel were resuspended in expansion or EGF-induction media in ultra-low
attachment plate for lentiviral infection. The filtered media containing lentivirus was added at a 1:1 (v/v) dilution to target
hepatocyte organoids. A second infection was performed at 60 hours post transfection. GFP expression was visualized at 48 hours
after the first infection. For 2D visualization of hepatocytes, hepatocytes were transitioned to geltrex-coated 2D culture in
expansion media (2 % v/v FBS) at day 5 post second infection, and imaged 2 days later on a Leica DMI 600B microscope.

FACS sorted NK1-R⁻ and NK1-R⁺ cells were used to derive NK1-R^low and NK1-R^hi, organoids, respectively (Figure S3A–D). NK1-R^low and NK1-R^hi organoids were dissociated into single cells and transduced with lentiviral constructs containing pLKO.1-puro-CMV empty vector and pLKO.1-puro-CMV-TurboGFP (Sigma), respectively, as per manufacturer’s protocol. NK1-R^low and NK1-R^hi/GFP organoids were plated as single cells at a 20:1 ratio (5×10³ total cells/well) and imaged every 4 hours for 4 days in a microscope incubation chamber. NK1-R^low and NK1-R^hi/GFP were plated at a 1:1 ratio (2×10⁴ total cells/well) and subject to flow cytometry analysis at several time points.