Despite significant advances in microfluidic technologies, sensitive single-cell RNA sequencing workflows that generate full-length cDNA remain plate-based. Miniaturization of these plate-based workflows has been increasingly implemented to reduce costs and increase throughput. We introduce a miniaturized, high-throughput workflow in which single HEK293FT cells were sorted by the f.sight™ into 384-well PCR plates, then libraries were prepared with the I.DOT.
Using this workflow, the SMART-seq and NGS library prep protocols, including cDNA amplification, tagmentation and library indexing, were performed at 10-fold reduced reaction volumes. Cells were lysed in volumes as low as 1 μL thanks to the f.sight’s precise cell dispensing. Furthermore, the I.DOT Liquid Handler's low-volume dispensing enabled the miniaturization of the library prep.
We found that the cDNA concentrations correlated with the cell size, which can aid when selecting the optimal number of PCR cycles in cDNA amplification for a given sample. This miniaturized workflow generated sufficient cDNA for tagmentation, and the cDNA and tagmented cDNA had the expected size distribution. There were 125 cells sequenced and more than 94% of the reads mapped to the human reference genome. Additionally, nearly 10,000 genes per single cell were detected at 1 million reads per cell. Using the f.sight and I.DOT combination to create a workflow that is readily compatible with standard SMART-seq protocols also gives one the flexibility to establish other plate-based library prep protocols.
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