Common cell lysis procedures distort ribosome profiling analyses of gene expression

Ribosome profiling is a powerful technique used to study gene expression on a transcriptome-wide scale. It involves sequencing of mRNA fragments protected by ribosomes from ribonuclease digestion. The initial steps commonly involve cell lysis followed by centrifugation and ribonuclease digestion. We find that centrifugation depletes 329 translated mRNAs in HEK293T cells. Many of these mRNAs encode cytoskeleton proteins. This suggests that the expression of a subset of mRNAs may be significantly underestimated in most ribosome profiling experiments. We show that omitting the centrifugation step after cell lysis can resolve this issue. Overall design: We investigated how sample preparation protocols affect Riboseq perfomance. This include the effect of Triton-X100 concentration in cell lysis buffer and the effect of lysate centrifugation.

核糖体图谱分析（Ribosome profiling）是一种可在转录组范围内开展基因表达研究的高效技术。该技术通过对被核糖体保护、免受核糖核酸酶降解的mRNA片段进行测序来实现研究目的。常规实验的初始步骤通常包括细胞裂解，随后依次进行离心与核糖核酸酶消化。本研究发现，在HEK293T细胞中，离心步骤会导致329种翻译中的mRNA出现损耗。其中多数mRNA编码细胞骨架蛋白。这提示，在多数核糖体图谱分析实验中，一类特定mRNA子集的表达水平可能被显著低估。我们证实，省略细胞裂解后的离心步骤可有效解决该问题。总体实验设计：本研究探究了样品制备方案对Ribo-seq实验性能的影响，具体涵盖细胞裂解缓冲液中Triton-X100浓度的作用，以及裂解液离心步骤的影响。