GSE168716
An internally normalized approach to comparing RNA levels between samples using nucleoside recoding chemistry
RNA sequencing allows for transcriptome-wide comparative studies of RNA levels, including gene expression, localization in subcellular compartments, or content of ribonucleoprotein complexes. It is often challenging, however, to separate real biological variation from technical artifacts arising from variable sample preparation, uneven contamination and difficulties normalizing sequencing counts between samples. These challenges are magnified in complex biochemical preparations, such as isolating polysomes to study translation. To address these challenges, we developed TILAC, an approach to compare RNA content between samples with internal controls and normalization. TILAC uses two different metabolic labels (4-thiouridine, s4U, and 6-thioguanisine, s6G) to differentially label RNA from each condition, allowing the samples to be pooled prior to downstream processing. TILAC uses nucleotide-recoding chemistry and sequencing to determine which RNAs are enriched in each sample. TILAC accurately identifies known changes in the transcriptome during RNA polymerase II inhibition and heat shock response. Using TILAC, we discovered a set of transcripts that are enriched in actively-translating ribosome complexes during stress, including MCM2 and DDX5, and verified their translational upregulation. These results demonstrate the power of TILAC to uncover differences between samples revealing new biology. Overall design: RNA isolated from Drosophila S2 cells +/- heat shock and +/- s4U or s6G, and from 293t cells +/- flavopiridol inhibitor and +/- s4U or s6G or +/- sodium arsenite and +/- s4U or s6G. RNA was also isolated from 293t cells +/- sodium arsenite and +/- s4U or s6G whose cell lysate was treated with +/- puromycin. RNA was subjected to oxidative-nucleophilic-aromatic-substitution chemistry. Sequencing libraries were prepared from all RNA or polysome-associated RNA.