DepleteX® Mito DNA Depletion Kit KIT2002 For post-library depletion of Illumina NGS libraries Contact Us If you have any questions, contact Technical Support at [email protected] Find us at our website: jumpcodegenomics.com Call us at 1.619.900.1701 DepleteX Mito DNA Depletion Kit v1.1 | May, 2024 © Copyright 2024, Jumpcode Genomics, Inc.; all rights reserved

The DepleteX* Mito DNA Depletion Kit targets DNA molecules derived from the human mitochondrial chromosome for removal from next generation sequencing (NGS) libraries. The kit leverages the CRISPR Cas9 endonuclease and its associated guide RNA to target and cleave human mitochondrial sequences. In samples with high mitochondrial content, such as ATAC-Seq libraries, use of the DepleteX Mito DNA Depletion Kit maximizes the re-assignment of sequencing reads to informative nuclear content and increases sequencing coverage of DNA derived from the nuclear fraction. * Human RNA NGS libraries with abundant...

Workflow DepleteX Mito DNA Depletion Kit (Post-Library Depletion) Step A: Depletion of Mitochondrial DNA □ Hands-ontime:~30 minutes | Totaltime:~2 hours 1. RNP Complex Formation & CRISPR Digestion 2. Bead Size Selection ® Safe Stopping Point D Hands-ontime:~30 minutes | Totaltime:~l hour ^ Safe Stopping Point DepleteX® Mito DNA Depletion Kit v1.1 | May, 2024 © Copyright 2024, Jumpcode Genomics, Inc.; all rights reserved | For research use only.

The DepleteX Mito DNA Depletion Kit contains enough material to deplete 24 NGS libraries. The kit contents and storage temperatures are indicated in the tables below. DepleteX® Mito DNA Depletion Kit v1.1 | May, 2024 © Copyright 2024, Jumpcode Genomics, Inc.; all rights reserved |

Required Materials and Equipment Type DepleteX® Mito DNA Depletion Kit v1.1 | May, 2024 © Copyright 2024, Jumpcode Genomics, Inc.; all rights reserved |

USERMANUAL v1.1Best PracticesGeneral • Periodically, optimizations and revisions are made to the kit components and user manual. Therefore, following the protocol included with or appropriate for the kit in question is important. This can be done by comparing the name and version number of the product to the name and version number of the protocol. If you need further assistance in this regard, please contact [email protected] • Use properly calibrated pipettes as library preparation is sensitive to pipetting error. • Follow good laboratory practices throughout the protocol, including...

Input Material Please review and follow the guidelines below for preparing DNA-Seq libraries that are optimal for depletion: The DepleteX Mito DNA Depletion Kit is optimized for 10 ng of NGS library material. Efficient depletion rates and high-quality NGS data have been validated with the use of the NEBNext® Ultra II FS DNA Library Prep Kit (Cat# E7805). The DepleteX Mito DNA Depletion Kit can also be used to deplete mitochondrial sequences from RNA-Seq libraries that contain a high proportion of transcripts derived from the mitochondrial genome. Since depletion occurs downstream of library preparation,...

□ Hands-ontime:~ 30 min | Total time:~2 hours Reagent Preparation 6. Mix gently by flicking the tube or pipetting up and down slowly. Collect the contents at the bottom of the tube by briefly spinning the tube in a microcentrifuge. DepleteX® Mito DNA Depletion Kit v1.1 | May, 2024 © Copyright 2024, Jumpcode Genomics, Inc.; all rights reserved | For research use only.

7. Place the tube in a thermal cycler with a heated lid set to >80°C. Run the following program: 8. Following incubation, proceed to the next step (Bead Size Selection). A2: Bead Size Selection 1. Add 30 pi of Nuclease-Free Water to the CRISPR digestion reaction from Step A1.7. 2. Add 40 pL (0.8X) of resuspended AMPure XP beads to the reaction. Mix well by pipetting up and down. Collect the contents at the bottom of the tube by briefly spinning the tube in a microcentrifuge. 3. Incubate at room temperature for 10 minutes. Mix the sample halfway through the incubation by pipetting up and down....

USERMANUAL v1.1 Step B: PCR Amplification □ Hands-ontime:~30min | Total time: ~1 hour Reagent Preparation *The number of PCR cycles may have to adjusted as yield will depend on sample type, library quality and mitochondrial content. 3. Proceed immediately to the next step (Bead Cleanup). 1. Add 80 pi (0.8X) of resuspended AMPure XP beads to the reaction. Mix well by pipetting up and down. Collect the contents at the bottom of the tube by briefly spinning the tube in a microcentrifuge. 2. Incubate the sample at room temperature for 10 minutes. Mix the sample halfway through the incubation by pipetting...

USER MANUAL v1.1 7. Open the cap of the tube while it is on the magnetic stand and allow the beads to air dry for up to 5 minutes. 8. Remove the tube from the magnetic stand and add 30 μL of Tris Buffer to the beads. Mix well to resuspend the beads by pipetting up and down. Collect the contents at the bottom of the tube by briefly spinning the tube in a microcentrifuge. 9. Incubate the sample at room temperature for 10 minutes. Mix the sample halfway through the incubation by pipetting up and down. Collect the contents at the bottom of the tube by briefly spinning the tube in a microcentrifuge....