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Miniaturization of Ribosomal RNA Depletion and Total RNA Library Preparation in Single Cells.

Miniaturization of library preparation methods has been shown to significantly decrease both costs and labor associated with next generation DNA sequencing, without sacrificing the quality of data obtained. By reducing reaction volumes, the number of samples prepared per kit increases, making this an approach that should be utilized among all prep types. Here we show that single-cell ribosomal RNA depletion and cDNA library preparation can be efficiently scaled to yield hundreds of high-complexity libraries per run, from both cell lines and dissociated tissues. We used the STP Labtech Mosquito HV to perform a miniaturized single cell total RNA prep with the Takara Bio SMART-Seq Stranded Kit on cells flow sorted into a 384 well plate. We observed no performance degradation when the protocol was scaled 4x, from 96 to 384 single cells per plate. The use of ribosomal RNA depletion, rather than poly-A selection, allows for a comprehensive, whole- transcriptome analysis of each individual cell. In summary, our results demonstrate a flexible approach for optimizing and miniaturizing challenging preparations to yield highly complex libraries.