DepleteXTM Nasopharyngeal Microbial RNA Boost Kit (24 Samples)
DepleteXTM Nasopharyngeal Microbial RNA Boost Kit (24 Samples)
The DepleteX™ Nasopharyngeal Microbial RNA Boost Kit (KIT1039) is designed to prepare RNA libraries by depleting human and bacterial rRNA and abundant human nasopharyngeal mRNA for sequencing on Illumina® instruments. It works with the CRISPRclean™ Plus Stranded Total RNA Prep with rRNA Depletion Kit (KIT1016) and processes samples to a fully depleted NGS library in under two days. A CRISPRclean Unique Dual Index Adapter Plate, sold separately, is required for high-throughput sequencing.
- Assay time: 13 hours
- Hands-on time: 4.5 hours
- Input RNA quantity: 5 ng to 100 ng
- Strand specificity: > 98% directional
- Multiplexing: Up to 96 Unique Dual Indexes
- Compatibility: Illumina® short read sequencing instruments
- Primary use: Detection of respiratory viruses with RNA genomes
The library preparation involves eight main steps: RNA fragmentation, first and second strand synthesis, adenylation, adapter ligation, PCR amplification, depletion, and final PCR amplification. Optional RNA fragmentation and conversion to double-stranded cDNA are included, with depletion using CRISPR Cas9. Post-depletion PCR amplification ensures only sequences of interest are amplified.
The kit includes reagents for 24 RNA-Seq libraries, with a shelf life of six months when stored properly. Key components include CRISPRclean Fragmentation Buffer Mix, First and Second Strand Synthesis Mixes, Adenylation Mix, Ligase Mix, Cas9, and Guide RNA, stored at specified temperatures.
- Total RNA
- CRISPRclean Unique Dual Index Adapter Plate
- AMPure® XP beads
- Various pipettes and RNase-free consumables
- Agilent 2100 Bioanalyzer® System
- Magnetic stand, microcentrifuge, thermal cycler, vortex, and ice
- Do not use the kit beyond six months from manufacturing.
- Maintain laboratory temperature between 20°–25°C.
- Ensure all consumables are RNase-free.
- Use properly calibrated pipettes.
- Do not mix plates by vortexing to avoid cross-contamination.
The protocol is optimized for 5 ng to 100 ng of total RNA from human nasopharyngeal samples. RNA should be free of genomic DNA and measured accurately. High-quality RNA with an RIN > 7 is recommended for optimal results.
Step A: RNA Fragmentation
Combine total RNA with CRISPRclean Fragmentation Buffer Mix and incubate in a thermal cycler based on RNA Integrity Number (RIN). Fragmentation is not recommended for RIN < 3.
- Step B: First Strand Synthesis - Combine fragmented RNA with CRISPRclean First Strand Synthesis Mix and Reverse Transcriptase, followed by thermal cycling.
- Step C: Second Strand Synthesis - Combine first strand synthesis product with Second Strand Synthesis Mix, followed by incubation and cleanup using magnetic beads and ethanol washes.
- Step D: Adenylation - Adenylate the second strand synthesis product using Adenylation Mix and Enzyme, followed by thermal cycling.
- Step E: Adapter Ligation - Ligate adapters to adenylated DNA, followed by cleanup with magnetic beads and ethanol washes.
- Step F: PCR Amplification - Amplify adapter-ligated DNA using PCR Primer and Master Mix, followed by cleanup.
- Step G: Depletion - Use CRISPRclean Guide RNAs and Cas9 for depletion of unwanted RNA, followed by cleanup.
Ensure all components are centrifuged before use, avoid excess material on pipette tips, and use freshly prepared ethanol for washes. The procedure can be paused at certain points for storage at -20°C.
Handle viscous components carefully and pipette precisely to avoid shortages. Adapters should not be premixed to prevent dimer formation. Guide RNA stocks should be returned to -80°C immediately after use.
The manual includes a comprehensive list of P7 and P5 index sequences, along with their reverse complements, for use with Illumina® sequencing platforms. These sequences are fully color balanced to avoid laser color complexity issues during sequencing.
Users are directed to the Illumina® website for the latest guidelines, software, and training recommendations for compatible instruments. This ensures proper handling and sequencing of the samples.
The product is covered by patents, trademarks, and copyrights owned by Jumpcode Genomics, Inc. Users may need to obtain additional third-party intellectual property rights for certain applications.
The document mentions several registered trademarks, including AMPURE®, BIOANALYZER®, ILLUMINA®, CRISPRclean™, and DepleteX™, highlighting the proprietary technologies involved.
Catalog excerpts
USER MANUAL 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 KIT1039 DepleteX™ Nasopharyngeal Microbial RNA Boost Kit (24 Samples) | December, 2023 © Copyright 2021, Jumpcode Genomics, Inc.; all rights reserved | For research use
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The DepleteX™ Nasopharyngeal Microbial RNA Boost Kit (KIT1039) used inconjuction with the CRISPRclean™ Plus Stranded Total RNA Prep with rRNA Depletion Kit (KIT1016) is designed to prepare strand-specific RNA libraries depleted of human and bacterial ribosomal RNA (rRNA) and abundant human nasopharyngeal mRNA for sequencing on Illumina® instruments. The protocol is meant for total cellular RNA isolated from nasopharyngeal samples. Protocol time is less than 2 days. This kit contains the reagents required to process a nasopharyngeal sample from total RNA through to fully depleted NGS library....
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USER MANUAL Second Strand Synthesis Safe Stopping Point Adapter Ligation Human RNA Depletion workflow The streamlined workflow for library preparation from total RNA involves eight main steps: RNA fragmentation, first strand synthesis, second strand synthesis, adenylation, adapter ligation, PCR amplification, depletion and final PCR amplification. The workflow begins with optional fragmentation of the RNA at high temperature. This is followed by first and second strand synthesis, which convert the RNA fragments to double-stranded cDNA. > 98% strand specificity is achieved through incorporation...
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The The DepleteX™ Nasopharyngeal Microbial RNA Boost Kit (KIT1039) used inconjuction with the CRISPRclean™ Plus Stranded Total RNA Prep with rRNA Depletion Kit (KIT1016) contains enough material to prepare twenty-four RNA-Seq libraries for Illumina® compatible sequencing. The shelf life of all reagents is six months from the date of manufacturing when stored properly. The kit contains the following reagents to be stored at the temperatures indicated in the table below: The CRISPRclean Unique Dual Index Adapter Plate for RNA Prep (KIT1017) (sold separately) is also required for library preparation...
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Kit contents Storage temp CRISPRclean Library Prep Cleanup Beads 4°C Required materials provided by the user Reagents • CRISPRclean Unique Dual Index Adapter Plate for RNA Prep (Set A): KIT1017 • AMPure® XP beads (stored at 4°C) • 80% Ethanol (freshly prepared and stored at room temperature) • RNase-free barrier pipette tips • Nuclease-free 1.5 mL microcentrifuge tubes • Thin-walled nuclease-free PCR tubes (Eppendorf™ LoBind) or similar • 96 well PCR plate non-skirted (Phenix Research™, # MPS-499) or similar • Adhesive PCR plate seal (BioRad®, # MSB1001) • Agilent 2100 Bioanalyzer® System RNA...
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USER MANUAL Warnings and precautions • We strongly recommend reading the following warnings and precautions. Periodically, optimizations and revisions are made to the components and manual. Therefore, it is important to follow the protocol included with the kit. If you need further assistance, contact [email protected]. • Do not use the kit beyond 6 months from the date of manufacturing. • The CRISPRclean First Strand Synthesis Mix may appear yellow in color. • The CRISPRclean® Plus Stranded Total RNA Prep with Microbial RNA Boost (Nasopharyngeal) Kit is intended to be used with the...
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V1.0 December 2023 Product launch KIT1039 DepleteX™ Nasopharyngeal Microbial RNA Boost Kit (24 Samples) | December, 2023 © Copyright 2021, Jumpcode Genomics, Inc.; all rights reserved | For research use only.
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USER MANUAL Library prep setup Starting materials The CRISPRclean® Plus Stranded Total RNA Prep with DepleteX™ Nasopharyngeal Microbial RNA Boost has been optimized and validated for 5 ng to 100 ng of total RNA isolated from human nasopharyngeal samples that contain a mix of human and bacterial content. Before beginning the protocol, total RNA is required to be free of contaminating genomic DNA. Treat the samples as recommended in RNA isolation protocols with RNase-free DNase. Resuspend and dilute RNA in RNase-free molecular biology grade water. Measure RNA concentration with a fluorometric method...
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□ Hands-on time: 10 min | Total time: 25 min Materials provided (brown) - CRISPRclean Fragmentation Buffer Mix (Z) (white or clear) - CRISPRclean Nuclease-free Water Required materials provided by the user • Total RNA • Nuclease-free microcentrifuge tube or plate • Thermal cycler NOTE: This protocol requires prior isolation of RNA through standard methods. Fragmentation times are dependent on the RIN. The RIN of the RNA sample must be determined with an Agilent Bioanalyzer® 2100 instrument or equivalent before starting library preparation. 1. For each reaction, combine the following reagents...
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□ Hands-on time: 10 min | Total time: 45 min (red) CRISPRclean First Strand Synthesis Mix (red) CRISPRclean Reverse Transcriptase • Fragmented RNA (from Step A) • Thermal cycler NOTE: Due to the viscosity of certain materials, preparing more than the stated number of reactions may result in a shortage of materials. All CRISPRclean enzyme components must be centrifuged at 600 x g for 5 seconds before opening the tube(s). Pipette only the necessary volume. Avoid excess material on the exterior of the pipette tip to ensure sufficient components for the stated number of reactions in the kit. 1. For...
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Materials provided (white or clear) - CRISPRclean Second Strand Synthesis Mix (white or clear) - CRISPRclean Resuspension Buffer (white or clear) - CRISPRclean Library Prep Cleanup Beads (room temp) Required materials provided by the user • First strand synthesis product (from Step B) • Thermal cycler • Adhesive PCR plate seal • 80% Ethanol, freshly prepared (room temp) • Magnetic stand 1. For each reaction combine the following on ice in a nuclease-free PCR tube or 96-well plate: 2. Mix thoroughly by pipetting up and down. 3. Program a thermal cycler as follows: 4. Incubate on the thermal cycler...
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