DepleteXTM Nasopharyngeal Microbial RNA Boost Kit (24 Samples)
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USER MANUAL Contact Us If you have any questions, contact Technical Support at support@jumpcodegenomics.com 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

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...

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...

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...

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...

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 support@jumpcodegenomics.com. • 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...

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.

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...

□ 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...

□ 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....

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...