High-Throughput Translational Profiling with riboPLATE-seq

Protein synthesis is dysregulated in many diseases, but we lack a systems-level picture of how signaling molecules and RNA binding proteins interact with the translational machinery, largely due to technological limitations. Here we present riboPLATE-seq, a scalable method for generating paired libraries of ribosome-associated and total mRNA. As an extension of the PLATE-seq protocol, riboPLATE-seq utilizes barcoded primers for pooled library preparation, but additionally leverages rRNA immunoprecipitation on whole polysomes to measure ribosome association (RA). We demonstrate the performance of riboPLATE-seq and its utility in detecting translational alterations induced by inhibition of protein kinases. Overall design: 96 paired riboPLATE- and PLATE-seq libraries, corresponding to independent TS543 glioma neurosphere samples seeded on a 96-well plate, plus 13 RNA-seq and 10 ribosome profiling libraries. 6 replicates per drug treatment (PP242, MNK-i1, BKM120, PP242+BKM120, PP242+MNK-i1, BKM120+MNK-i1, AZD8055, 4EGi-1) and 48 DMSO-treated controls, plus three replicates each of ribosome profiling and RNA sequencing for PP242 or DMSO treatment at 30 minutes, two further replicates at 6 hours PP242 or DMSO treatment, and one additional unpaired 6-hour DMSO-treated RNA-seq library.