RT Journal Article SR Electronic T1 Polysome-profiling in small tissue samples JF bioRxiv FD Cold Spring Harbor Laboratory SP 104596 DO 10.1101/104596 A1 Shuo Liang A1 Hermano Bellato A1 Julie Lorent A1 Fernanda Lupinacci A1 Vincent Van Hoef A1 Laia Masvidal A1 Glaucia Hajj A1 Ola Larsson YR 2017 UL http://biorxiv.org/content/early/2017/02/01/104596.abstract AB Polysome-profiling is commonly used to study genome wide patterns of translational efficiency, i.e. the translatome. The standard approach for collecting efficiently translated polysome-associated RNA results in laborious extraction of RNA from a large volume spread across multiple fractions. This property makes polysome-profiling inconvenient for larger experimental designs or samples with low RNA amounts such as primary cells or frozen tissues. To address this we optimized a non-linear sucrose gradient which reproducibly enriches for mRNAs associated with >3 ribosomes in only one or two fractions, thereby reducing sample handling 5-10 fold. The technique can be applied to cells and frozen tissue samples from biobanks, and generates RNA with a quality reflecting the starting material. When coupled with smart-seq2, a single-cell RNA sequencing technique, translatomes from small tissue samples can be obtained. Translatomes acquired using optimized non-linear gradients are very similar to those obtained when applying linear gradients. Polysome-profiling using optimized nonlinear gradients in HCT-116 cells with or without p53 identified a translatome associated with p53 status under serum starvation. Thus, here we present a polysome-profiling technique applicable to larger studies, primary cells where RNA amount is low and frozen tissue samples.