RNA sequencing (RNA-seq) is widely used to perform expression analysis of entire transcriptomes with high sensitivity and a wide dynamic range. Despite being a pivotal tool in biomarker discovery, RNA-seq can be challenging in cases where input amounts are low or comprised of degraded RNA samples. This is common when working with patient-derived samples such as tissue biopsies, blood, or other biofluids.
The SMARTer Stranded Total RNA-Seq Kit v3 – Pico Input Mammalian (referred to as “Pico v3”) is designed for efficient preparation of sequencing libraries from picogram amounts (250 pg–10 ng) of high- or low-quality total RNA. Reverse transcription (RT) via random priming makes the kit suitable for RNA of any quality (RIN > 2–10, DV200 > 25%) including highly degraded FFPE and laser capture microdissection (LCM) samples or cell-free RNA (cfRNA) from biofluids, serum, plasma, exosomes, etc.
Unlike most oligo(dT) primed approaches, the random priming approach used by Pico v3 allows for whole transcriptome profiling of coding and noncoding RNA biotypes (e.g. mRNA, lncRNAs, lincRNAs, etc.). Compared to approaches that only employ end-counting, the SMART® (Switching Mechanism at 5’ end of RNA Template) cDNA synthesis technology provides full gene body coverage for information on isoforms, fusions, etc. Furthermore, the directionality of the template-switching reaction preserves the strand orientation of the original RNA, making it possible to obtain strand-specific sequencing data from the synthesized cDNA.
The kit includes ZapR technology, a cas9-based post-library depletion technique to effectively deplete rRNA from picogram amounts of total RNA, unlike competitor depletion technologies that require significantly higher nanogram inputs. The workflow also incorporates unique molecular identifiers (UMIs) during reverse transcription to mitigate potential PCR errors and amplification biases, enabling quantitative analysis and accurate variant calling, especially for rare mutations.
Biomarker discovery also benefits from faster sequencing turnaround times. Singular Genomics has developed an innovative benchtop sequencer, the G4 Sequencing Platform, that leverages a 4-color rapid sequencing by synthesis (SBS) chemistry with advanced optics and fluidics engineering to provide single-day turnaround times across all applications. By combining fast run times and the ability to run up to 4 flow cells, with 16 independently addressable lanes, the G4 delivers accuracy, speed, power, and unprecedented flexibility for a wide range of genomic applications.
Here we show that total RNA libraries prepared with Pico V3 from 10 ng or 0.25 ng of Human Brain Total RNA Control yields similar sequencing metrics and data quality on the Singular Genomics G4 sequencing platform as on the industry-standard sequencing platform. These results show that researchers now have access to a fast, accurate, and cost-effective solution to power biomarker discovery from challenging and low input samples.