DepleteX® Rare Transcript Boost Kit KIT1020 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 Rare Transcript Boost Kit v1.1 | April, 2024 © Copyright 2024, Jumpcode Genomics, Inc.; all rights reserved

The DepleteX® Rare Transcript Boost Kit removes the highest expressing transcripts from RNA-Seq libraries prior to sequencing, allowing the user to better detect less abundant transcripts and rare isoforms. The kit leverages the CRISPR Cas9 endonuclease and associated guide RNA to remove abundant protein-coding transcripts (~70% of total sequencing reads in next-generation sequencing (NGS) data) from poly(A)-selected RNA-Seq libraries prepared from fibroblasts and whole blood. DepleteX® Rare Transcript Boost Kit v1.1 | April, 2024 © Copyright 2024, Jumpcode Genomics, Inc.; all rights reserved...

Workflow The protocol involves a 16-hour incubation for effective depletion. We recommend beginning the protocol at the end of the workday on Day 1 and continuing with the remaining portion of the protocol on the morning of Day 2. DepleteX™ Rare Transcript Boost Kit (Post-Library Depletion) Step A: Depletion of Highly Expressed RNA Hands-on time: ~30 min | Total time: ~17 hours (including 16-hour incubation) 1. RNP Complex Formation & CRISPR Digestion 2. Bead Size Selection Safe Stopping Point Step B: PCR Amplification Hands-on time: ~30 min | Total time: ~1 hour 1. PCR Amplification 2. Bead...

The DepleteX Rare Transcript Boost Kit contains enough material to deplete 24 RNA-Seq libraries. The kit contents and storage temperatures are indicated in the tables below. DepleteX® Rare Transcript Boost Kit v1.1 | April, 2024 © Copyright 2024, Jumpcode Genomics, Inc.; all rights reserved |

Required Materials and Equipment Type DepleteX® Rare Transcript Boost Kit v1.1 | April, 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] • This protocol describes the reagents, best practices, workflow, and method details for DepleteX Rare Transcript Boost kit. • Use properly calibrated pipettes...

Please review and follow the guidelines below for preparing RNA-Seq libraries that are optimal for depletion: This product is designed for optimal performance with RNA-Seq libraries prepared from human fibroblast or whole blood. Ribosomal RNA depletion is not included with this product. We recommend the use of poly(A)-selected libraries to ensure that little or no rRNA remains in the library when depletion is performed. The Jumpcode depletion protocol works most effectively with RNA-Seq libraries of > 420 bp fragment sizes. For many library preparation methods involving RNA fragmentation by heat,...

Before attempting this protocol for the first time, it is highly recommended that the user read the "Input Material" section on page 7. The protocol includes an overnight (16 hours) incubation step. We recommend starting the protocol at the end of the workday on Day 1 and complete the procedure on Day 2. Reagent Preparation DepleteX® Rare Transcript Boost Kit v1.1 | April, 2024 © Copyright 2024, Jumpcode Genomics, Inc.; all rights reserved | For research use only.

Total Volume 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. 7. Place the tube in a thermal cycler with a heated lid set to >80°C. Run the following program: NOTE: CRISPR digestion for 16-hours is an overnight incubation step. The protocol is designed so that if digestion is begun in the evening day 1, the sample can be removed from the thermal cycler the next morning on day 2 and proceed with the remainder of the protocol. Use of a water bath is not recommended for this incubation...

USERMANUAL v1.1 Step B: PCR Amplification Reagent Preparation *The number of PCR cycles after depletion correlates inversely with DNA input. With the recommended input of 10 ng and 10 PCR cycles, a final total library yield of 100-300 ng is expected. 3. Proceed immediately to the next step (Bead Cleanup). 1. Add 60 pi (0.6X) 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...

USER MANUAL v1.1 5. Repeat the previous wash step. 6. Briefly spin the tube, place it back on the magnetic stand. Ensure that all residual ethanol is removed at this step. 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...