JUMPCCDDE GENOMICS CRISPRclean™ Plus Stranded Total RNA Prep with rRNA Depletion(Human, Mouse, Rat, Pan Bacteria)CRISPR-based ribodepletion strategy optimized with stranded RNA prep increases coverage of lower-expressing transcripts from complex biological samples. Highlights • Streamlined CRISPRclean workflow: stranded total RNA library prep with ribodepletion • Ideal for analysis of samples containing complex mixtures of eukaryotic and bacterial rRNA • 3 to 7-fold increase in coverage of SARS-CoV-2 and other genomes in nasopharyngeal (NSP) samples • Single workflow to detect viral genomic data, microbiome composition, co-infections, and host gene expression Introduction The unbiased nature of shotgun metagenomic and metatranscriptomic sequencing has unlimited potential in the discovery and analysis of organisms co-existing in diverse biological systems (example: wastewater surveillance to profile viral genetic diversity across infected communities). In this datasheet nasopharyngeal swabs from clinically COVID positive patients were used to show how CRISPRclean Plus Stranded Total RNA Prep with rRNA Depletion (Human, Mouse, Rat, Pan Bacteria) can be applied to track viral spread during a pandemic. Much of the transcriptomic data captured is uninformative, due to the abundance of ribosomal RNA (rRNA) that interferes with efficient identification of biologically significant transcripts. CRISPRclean technology harnesses the power of the CRISPR system to deplete rRNA sequences from biological samples containing mixtures of mammalian, bacteria, and pathogenic agents. CRISPRclean Plus Stranded Total RNA Prep with rRNA Depletion is an optimized, workflow that simultaneously detects viral genomic data, microbiome composition, co-infections, and host gene expression. CRISPRclean Plus Stranded Total RNA Prep with rRNA Depletion reassigns sequencing reads from abundant molecules to higher value and lower expressing transcripts to understand gene expression of changing environmental conditions. In this datasheet we use SARS-CoV-2 (COVID) as a proxy for detection of RNA viruses in complex sample types containing eukaryotic, bacterial, and viral sequences. The adaptation of the revolutionary CRISPR technology to NGS library construction enables the process for discovery in shotgun metagenomic and metatranscriptomic sequencing. Workflow Optimized for 9 hours assay time with 3.5 hours hands-on time from total RNA to sequencing ready libraries. >98% strand specificity is achieved through incorporation of dUTP during second strand synthesis. The innovative step is the CRISPR-powered depletion of bacterial, human, mouse, or rat rRNA sequences in adapter ligated libraries. CRISPR-powered depletion is performed in two successive incubations, the first to cleave bacterial rRNAs and the second to cleave eukaryotic rRNAs. Cas9 and guide RNAs are combined to form the ribonucleoprotein complex specifically programmed to cut specific rRNA sequences. Cleaved rRNA sequences cannot be amplified, and are removed by size selection with magnetic beads. The final product is a directional and refined library ready for short read next-generation sequencing (NGS). CRISPRclean™ Plus Stranded Total RNA Prep with rRNA Depletion (Human, Mouse, Rat, Pan Bacteria) | October, 2021 © Copyright 2021, Jumpcode Genomics, Inc.; all rights reserved | For research use only.

First Strand Synthesis Second Strand Synthesis Adapter Ligation Hands-on time: 3.5 Hours | Assay time: 9 Hours Read Counts - Top 10 Organisms - Sample 404 - Ct 26.94 Figure 1: CRISPRclean Plus Stranded Total RNA Prep with rRNA Depletion (Human, Mouse, Rat, Pan Bacteria) is a streamlined workflow from total RNA to sequencing-ready, strand-specific libraries in 7 steps with multiple safe stopping points. Ideal for analysis of samples containing complex mixtures of organisms Bacterial rRNA Fold Change - Reads Per Million Depleted/Undepleted Correlation Of Depletion Replicates: Sample 391 Figure...

JUMPCCDDE GENOMICS Increased sensitivity to detect lower-expressing, biologically relevant transcriptsSingle workflow to detect viral genomic data, microbiome composition, co-infections, and host gene expression Sample Ct Table 1: Better RNA virus detection and strain calling results with CRISPRclean. COVID strain 20G was called only in the depleted sample S4 (Ct of 27.60). COVID presence was detected only in depleted samples, S5 and S6 samples at 32.25 and 33.45 Ct, respectively. CRISPRclean™ Plus Stranded Total RNA Prep with rRNA Depletion (Human, Mouse, Rat, Pan Bacteria) | October, 2021 ©...

Figure 7: Increased bacterial detection sensitivity with clinical NSP samples. Summary CRISPRclean Plus Stranded Total RNA Prep with rRNA Depletion is an optimized workflow that simultaneously detects viral genomic data, microbiome composition, co-infections, and host gene expression. CRISPRclean Plus Stranded Total RNA Prep with rRNA Depletion reassigns sequencing data from abundant molecules to higher value and lower expressing transcripts in order to understand gene expression of changing environmental conditions or treatments. In this datasheet we have presented data using nasopharyngeal...