Sample Processing Has Important Consequences For Single Cell Sequencing Studies

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Single-cell transcriptomics is a relatively new technology that reads and reports the gene expression profile of individual cells by simultaneously measuring the mRNA concentration of all the cell’s genes. This is achieved through microfluidic chip single cell isolation, barcoding, cDNA amplification and sequencing. Single cell resolution has the advantage that it can filter out contaminating cells in a population, for example normal tissue cells mistakenly captured in a tumor biopsy, or for the analysis of a specific cell type, such as a stem cell. It also potentially allows for very small sample sizes. This technology is potentially valuable for analyzing samples obtained through biobank studies, such as blood and others that contain cells.

When using biobanked (cryopreserved) samples, the processing of those samples needs to be considered. A recent preprint with senior author Holger Heyn of Centro Nacional de Análisis Genómico (CNAG-CRG), Barcelona, Spain, pointed out that sample processing after sample isolation is extremely important for valid single cell sequencing in order to obtain results that can be compared across studies, and even within studies. The authors compared room temperature processing with various time points, up to 48 hours, and also processing conducted exclusively at 4ºC. Such 4ºC processing, as is often employed in other single cell assay types, such as flow cytometry, was found to be ideal.

Peripheral blood mononuclear cells (PBMCs) from health donors and leukemic cells from cancer patients were processed/ stored at room temperature in a time series up to 8 hours, and including 24h and 48h timepoints. Following room temperature storage, the cells were cryopreserved and single cell sequenced. The results indicated that although the cells remained viable room temperature storage for greater than 2 hours had global effects on gene expression that increased with time.

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At time points over 2 hours at room temperature gene expression was globally down-regulated and the transcriptional signature exhibited characteristics of a cold-shock response. In addition, a pronounced downregulation of immune cell type specific genes was observed pointing to a loss of identity. The authors found that it was possible to classify affected cells using a cold-shock score, and to correct for global gene expression changes in silico, especially for samples stored at room temperature for less than 8 hours.

As the authors pointed out in silico correction is not ideal as it can homogenize cell populations and mask true biological phenomena. The authors therefore investigated the effect of immediately storing samples at 4ºC upon single cell sequencing profiles. When PBMC or leukemic samples were stored at 4ºC up to 48 hours before cryopreservation, no global gene expression artifacts were detected.

“The here detected artifacts are important to consider when planning single-cell experiments. Failing to select suitable samples or to correct datasets will lead to biased or false reporting,” concluded the authors.