PRJNA561907
Significant reduction of inter-cell variability with improved RNA protection in SCRB-seq
The realization of single cell RNA-seq has significantly expanded our ability to characterize different cell states and to identify rare cell phenotypes from heterogeneous populations. However, the robustness of such analysis requires highly reliable biochemical assays that do not introduce significant variations into the single cell quantitation. During analysis of the critical steps of the single cell RNA barcoding and sequencing (SCRB-seq) protocol, a powerful method for the identification of single cell transcriptomes, we found that a major limiting factor is the significant degradation of RNA resulting from the high temperatures required to inactivate proteinase K and adjust the reaction volume. We show that avoiding the usage of proteinase K and replacing with RNase inhibitor, leads to a great improvement of the long fragment cDNA and a 67% increase in the number of detected genes per cell. Notably, we find that this high-temperature induced degradation contributes to 25% of the single-cell transcriptional variability: the intrinsic inter-cell variability within a single cultured cell population reduced from 41% using SCRB-seq to 16% using improved protocol, mostly resulting from an enhanced detection of less highly expressed transcripts. Thus, by maintaining the integrity of the single cell RNA with this simple modification, we reduce the major source of the transcriptional noise otherwise present in the experiment, enabling a more accurate depiction of the differences and similarities between different individual cells, thus allowing a better detection of rare cells in heterogeneous populations.