Pbabe egfp

The open reading frame of Nrp1 was cloned into the pBabe vector. The open reading frame of Sema3a was cloned into pBabe-EGFP (Parker et al., 2012 (link)) (addgene, 36999). shNrp1 plasmids were purchased from Origene (OriGene, TG513573A). 15 µg of retroviral plasmid DNA was transfected using FuGENE HD (Roche, E2311) into Platinum E cells (Cell Biolabs), which were plated on a 10-cm tissue culture dish at a density of 5 × 10⁶ cells per dish, 24 h before transfection. After 48 h of transfection, viral medium was harvested and filtered through a 0.45 μm cellulose filter. The viral supernatant was concentrated overnight using Retro-X concentrator (Clontech, 631456) according to manufacturer’s protocol. The virus was then resuspended in growth medium mixed with Polybrene (Sigma) to a final concentration of 6 μg ml⁻¹.
Tw2-MB and Pax7-MB were plated at a density of 200,000 cells per 35-mm plate in growth medium. After 24 h, the growth medium was replaced with freshly made viral mixture containing Polybrene and bFGF (5 ng ml⁻¹). 24 hrs later, viral medium was replaced with growth medium with bFGF for recovery.