Library Preparation

We offer a variety of library preparation options depending on the input sample type and the research being performed. If you are unsure which library prep method is right for your project or require a different library prep method than listed below, please contact us to further discuss your options. We make extensive use of automation strategies during library prep to maintain consistent results during high-throughput workflows, and to support miniaturization of assays to reduce costs where possible.

mRNA

Our most commonly performed library preparation option uses TakaraBio's SMART-Seq v4 Ultra Low Input RNA Kit with Illumina's Nextera XT DNA Library Preparation Kit. The SMART-Seq v4 protocol is designed to generate high-quality, full-length cDNA directly from 1–1,000 cells or 10 pg–10 ng of high quality total RNA. This process relies on template switching activity of reverse transcriptases to enrich for full-length cDNAs and to add defined PCR adapters directly to both ends of the first-strand cDNA, ensuring the final cDNA libraries contain the 5′ end of the mRNA and maintain a true representation of the original mRNA transcripts. The cDNA is then passed through to the Nextera XT protocol where it is simultaneously fragmented and tagged with sequencing adapters in a single tube enzymatic reaction, then a bead-based library cleanup is performed.

Total RNA

The SMARTer Stranded Total RNA-Seq Kit - Pico Input Mammalian protocol is compatible with picogram-inputs of total RNA from high-quality or partially degraded samples, and works similarly to the above SMART-Seq v4 kit. However, the workflow used in this kit takes advantage of a novel technology allowing removal of ribosomal cDNA (cDNA fragments originating from rRNA molecules) after cDNA synthesis using probes specific to mammalian rRNA. The rRNA depletion method used in this kit makes it especially well-suited for working with very small quantities of total RNA.

miRNA

For miRNA library preparation, we use the QIAseq miRNA Library Kit from Qiagen. Total RNA is used as the starting material and adapters are ligated sequentially to the 3' and 5' ends of miRNAs in an unbiased reaction. Subsequently, universal cDNA synthesis with UMI assignment, cDNA cleanup, library amplification and library cleanup are performed. The modified oligonucleotides in this kit virtually eliminate the presence of adapter dimers in the sequencing while bead-based cleanups eliminate the majority of unwanted background noise. The UMIs allow the removal of amplification or sequencing artifacts to ensure that data analysis is focused on a true count of the original miRNA transcripts.