Category: Automation and High-Throughput Technologies
As the cost of sequencing has continued to decline by orders of magnitude in the past ten years, scientists are enabled to ask deeper and more complex questions about transcriptomics. Consequently, the variety, quantity, and demands of RNA sequencing experiments have all increased. The Clontech SMART-Seq® v4 Ultra® Low Input RNA Kit incorporates the SMART (Switching Mechanism at 5’ End of RNA Template) technology. This technology utilizes the template switching activity of reverse transcriptases to add PCR adapters directly to both the 5’ and 3’ ends of the first-strand cDNA before amplification using LD PCR. Here, we document information and results pertaining to the miniaturization of this process utilizing the Echo® 525 Liquid Handler, effectively reducing the reagent cost and processing time for the workflow, addressing current throughput needs of RNA sequencing. We found that miniaturization of SMART-Seq v4 can produce sufficient library for sequencing, across reaction sizes, with PCR cycling of 18. We show that varying reaction miniaturization and input RNA in this 18-cycle sample set did not produce any differentially expressed genes. We also confirmed that the variables of input RNA, amount of reaction miniaturization, and amount of PCR cycling are all interconnected. For example, low-input RNA experiments will require additional PCR cycling, especially if the reaction volume has been reduced significantly. Increasing sample RNA input or reaction volume will reduce the amount of PCR cycling necessary (and subsequent PCR bias), but the reagent cost savings will be less drastic. It is very much up to the user and the defined experiment to balance the cost-savings and throughput with the amplification bias in both SMART-Seq v4 and Nextera XT.
Jefferson Lai– Applications Scientist, Labcyte, Inc., San Jose, California
San Jose, California
Jefferson attended the University of California, Berkeley, for his undergraduate. He graduated with a BS in Microbial Biology and worked in Dr. Steve Lindow's lab developing a transposon mutagenesis screen for quorum sensing in Pseudomonas syringae. After, Jefferson began working in the synthetic biology industry, first at Amyris Inc for 3 years, then Intrexon Corp for 4 years, both as Associate Scientist, before joining Labcyte Inc as an Applications Scientist. At Amyris, Jefferson worked on replacing Saccharomyces cerevisiae native central metabolism with an augmented one to achieve yields greater than theoretically possible using native metabolism. This work has been published in Nature. At Intrexon, Jefferson developed genetic tools to engineer into a non-model host and metabolic engineering for production of a chemical, then developed, operationalized, and managed the in-house NGS pipeline. At Labcyte, Jefferson develops processes used in synthetic biology using the Labcyte Echo acoustic liquid handler.