DepleteX® Mito DNA Depletion Kit
DepleteX® Mito DNA Depletion Kit
The DepleteX® Mito DNA Depletion Kit is designed to remove human mitochondrial DNA from Illumina NGS libraries using CRISPR Cas9 technology. This process enhances the sequencing coverage of nuclear DNA by reducing mitochondrial content, which is particularly beneficial for samples like ATAC-Seq libraries.
- Total Assay Time: Approximately 3 hours
- Hands-on Time: Approximately 1 hour
- Samples Per Kit: 24
- Sample Type: Human DNA NGS Library
- DNA Library Input: 10 ng
- DNA Library Size: ≥ 200 bp
- Method: CRISPR-Cas9 mediated depletion
The workflow consists of two main steps: Depletion of Mitochondrial DNA and PCR Amplification. Each step includes specific sub-steps such as RNP Complex Formation, CRISPR Digestion, and Bead Size Selection for depletion, followed by PCR Amplification and Bead Cleanup.
The kit includes reagents for 24 reactions, stored at -20°C and -80°C. Key components include Cas9, RNase Inhibitor, and Guide RNA.
Essential materials include DNA LoBind Tubes, AMPure XP Beads, and a Qubit Fluorometer. Equipment needed includes pipettes, a vortex mixer, a microcentrifuge, and a PCR thermal cycler.
Ensure proper calibration of pipettes and use DNase- and RNase-free consumables. Follow the protocol version specific to the kit and handle reagents as per storage guidelines to maintain their efficacy.
The kit is optimized for 10 ng of NGS library material. It can also deplete mitochondrial sequences from RNA-Seq libraries. Accurate DNA quality assessment is recommended for optimal results.
Step A: Depletion of Mitochondrial DNA
Involves RNP Complex Formation and CRISPR Digestion, followed by Bead Size Selection to isolate the depleted DNA.
Step B: PCR Amplification
Involves amplifying the depleted DNA using specific primers and a thermal cycler.
The document outlines the procedures for using the DepleteX® Mito DNA Depletion Kit v1.1 by Jumpcode Genomics. It includes detailed steps for bead cleanup, which is crucial for preparing a depleted DNA library. The process involves adding AMPure XP beads, incubating, washing with ethanol, and resuspending in Tris Buffer. The final product is a depleted DNA library ready for sequencing.
- Add 80 μL of AMPure XP beads and mix well.
- Incubate at room temperature for 10 minutes, mixing halfway.
- Use a magnetic stand to clear the solution and discard the supernatant.
- Wash twice with 80% ethanol while on the magnetic stand.
- Air dry the beads, then resuspend in Tris Buffer.
- Incubate again and transfer the supernatant to a new tube for the depleted DNA library.
Jumpcode Genomics recommends assessing the library yield using a dsDNA-specific fluorescence-based method and checking library size with an Agilent 2100 Bioanalyzer before sequencing. If DNA fragments are smaller than 200 bp, a gel-based size selection is advised.
This product is intended for research purposes only and is not for therapeutic or diagnostic use in humans or animals. It is covered by patents and trademarks owned by Jumpcode Genomics, Inc. Users may need additional third-party intellectual property rights for certain applications.
For support, contact Jumpcode Genomics at [email protected] or call 1.619.900.1701.
Catalog excerpts
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
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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...
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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.
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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 |
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Required Materials and Equipment Type DepleteX® Mito DNA Depletion Kit v1.1 | May, 2024 © Copyright 2024, Jumpcode Genomics, Inc.; all rights reserved |
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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...
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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,...
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□ 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.
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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....
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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...
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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....
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