RiboTag-based Polyribosome Immunoprecipitation

Polyribosome immunoprecipitation was achieved as described in ^{14 (link), 33 (link)}. Briefly, pooled tissue from RiboTag (RT)^+/+ mice with virally mediated Cre expression in VP→MDT neurons (n= 4–5 mice per sample) was homogenized and 800-μL of the supernatant was incubated in HA-coupled magnetic beads (Invitrogen: 100.03D; Covance: MMS-101R) overnight at 4°C. Magnetic beads were then washed in high salt buffer. Following TRK lysis buffer addition, RNA was extracted with the RNeasy Micro kit (Qiagen: 74004). For input, 50 ng of RNA was used and 1–2 ng of RNA from immunoprecipitated samples were amplified using the Low RNA Input kit (NanoString Technologies®). All samples were then processed with the nCounter Master Kit (NanoString Technologies®) by UMSOM IGS on a custom-made gene expression Code set (see Supplementary Table 3 for primer sequences). Data were analyzed with nSolver Analysis software ^{14 (link)}.