Multiple freeze-thaw cycles impact on lead to a loss of consistency in poly(A)-enriched RNA sequencing

RNA-Seq is ubiquitous, but depending on the biological question or the sample source, some collection methods require variable or sub-optimal sample handling. Rare tissue may need to be fixed or frozen prior to library preparation, or samples may remain at room temperature while other samples or reagents are handled. These challenges create uncharacterized impacts on sample quality. Understanding the relevant experimental factors that impact sample quality is crucial to ensuring reproducible RNA-Seq results. Here, we tested the susceptibility of poly(A)-enriched RNA-Seq results after multiple freeze-thaw cycles. Despite its prevalence, the impact of freeze-thaw as a distinct mode of transcript degradation with distinct effects has not been comprehensively quantified, and has only been examined in the context of RIN. We assessed sample quality independently of RIN by simulating read count variability to capture the noise between technical replicates. Each additional freeze-thaw cycle increased the random counts between technical replicates by approximately 4%, leading to a complete negation of differential expression reproducibility after three freeze-thaw cycles . These effects are not captured by RIN. The use of poly(A)-enrichment for RNA sequencing is pervasive in library preparation of frozen tissue, and thus, it is important during experimental design and data analysis to consider the impact of repeated freeze-thaw cycles on reproducibility.