JUMPCCDDE GENOMICS CRISPRclean™ Stranded Total RNA Prep with rRNA Depletion(Human, Mouse, Rat)CRISPR-based ribodepletion strategy improves sensitivity for the detection of lower expressing transcripts from mammalian tissues Highlights • One-day workflow: 7.5 hours assay with 3 hours of hands-on time • Effective single tube, multi-species depletion with CRISPR-Cas9 mediated technology • Optimized library prep and depletion workflow generates high quality representation of transcripts • Consistent full-length, uniform transcript coverage • Increased sensitivity to detect lower expressing, biologically relevant transcripts Introduction RNA sequencing has unleashed tremendous data generation to profile gene expression of diverse mammalian cell systems. The challenge is to efficiently eliminate abundant or uninformative sequences, such as ribosomal RNA (rRNA), in order to shift discovery to the previously, or lower expressing, and biologically interesting transcripts. CRISPRclean Stranded Total RNA Prep with rRNA Depletion (Human, Mouse, Rat) harnesses the power of CRISPR-based depletion with stranded RNA library preparation to specifically remove human, mouse, and rat rRNA sequences from adapter ligated cDNA libraries. CRISPRclean Stranded Total RNA Prep with rRNA Depletion reassigns sequencing reads from abundant molecules to higher value and lower expressing transcripts in an unbiased manner without disturbing the relative transcript abundance or complexity of the library. CRISPRclean Stranded Total RNA Prep with rRNA Depletion improves discovery of high value, biologically interesting transcripts, including low-expressing transcripts. One-day workflow from RNA to sequencing ready library Library preparation and rRNA depletion is completed through 7 steps with multiple safe stopping points (Figure 1). Greater than 98% of strand specificity is RNA First Strand Second Strand . t Adapter Depletion PCR Fragmentation Synthesis Synthesis y Ligation of rRNA Amplification Q Hands-on time: ~3 Hours | Assay time: 7.5 Hours Figure 1: CRISPRclean Stranded Total RNA Prep with rRNA Depletion (Human, Mouse, Rat) is a simple and streamlined 1-day workflow from total RNA to sequencing-ready, strand-specific libraries in 7 steps with multiple safe stopping points. CRISPRclean™ Stranded Total RNA Prep with rRNA Depletion (Human, Mouse, Rat) | October, 2021 © Copyright 2021, Jumpcode Genomics, Inc.; all rights reserved | For research use only.

Methods Libraries were prepared from universal reference total RNA for human, mouse, rat, chicken, cow, and dog at different input amounts ranging from 5 ng to 100 ng. Libraries were generated with CRISPRclean Stranded Total RNA Prep with rRNA Depletion (Human, Mouse, Rat) and sequenced on an Illumina® NextSeq™ 2000 instrument at 64M reads and 2x150 bp paired end reads. Data analysis was performed using a range of bioinformatic tools such as the RSeQC for transcript coverage and uniformity, STAR aligner for human genome and ERCC alignments, and custom pipelines were used to assess rRNA depletion...

CRISPRclean Comparison Between 5 ng and 100 ng RNA Input Optimized library prep and depletion workflow generates high quality representation of transcripts CRISPRclean Replicate Performance - 5 ng Genes (Sorted Low to High Expression in 5 ng Library) 0 Figure 4: Robust and reproducible. High reproducibility of read counts in replicate depleted CRISPRclean libraries. Sequence read counts of ERCC control spiked into 5 ng of UHR RNA input displayed high correlation (R2 values for linear fit) between depleted library replicates using CRISPRclean. Human RNA Expression - CRISPRclean 5 vs 100 ng Input...

Gene detection across at 1X and 10X coverage Increased sensitivity toinputs detect lowerexpressing, biologically relevant transcripts directional RNA library preparation kit with a novel CRISPR-mediated depletion strategy that increases detection sensitivity of lower expressing and biologically relevant transcripts while generating consistent fulllength, uniform transcript coverage with minimal offtarget. Figure 8: Consistent gene detection at 10x coverage in depleted libraries. The number of genes is assessed at 10x coverage in libraries prepared with 5 ng, 25 ng, and 100 ng UHR RNA. Gene detection...