Mar 17, 2026
KAPA RNA HyperPrep Kit with RiboErase and HMR
This protocol is a draft, published without a DOI.
- Kristine Wylie1,
- Jane Schrimpf1,
- Madison Eschbach1,
- Hunter Olson1
- 1Washington University
Protocol Citation: Kristine Wylie, Jane Schrimpf, Madison Eschbach, Hunter Olson 2026. KAPA RNA HyperPrep Kit with RiboErase and HMR. protocols.io https://protocols.io/view/kapa-rna-hyperprep-kit-with-riboerase-and-hmr-jv9ecn93f
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: March 16, 2026
Last Modified: March 17, 2026
Protocol Integer ID: 313350
Keywords: roche kapa rna hyperprep kit with riboerase, rna hyperprep kit with riboerase, roche kapa rna hyperprep kit, rna hyperprep kit, rna sample, depleted rna, riboerase, globin mrna, rrna, hmr this protocol
Funders Acknowledgements:
NIH NCCIH
Grant ID: U01 AT012970
Abstract
This protocol was adapted from the Roche KAPA RNA HyperPrep Kit with RiboErase and HMR to deplete globin mRNA and rRNA (human and custom-bacterial) from RNA samples. Create cDNA from the depleted RNA and make libraries.
Materials
- KAPA Pure beads
- 80% ethanol in molecular biology grade water
- RNA samples
- Oligo Hybridization Master Mix components:
- Hybridization Buffer
- Globin Hybridization Oligos (HMR)
- Hybridization Oligos (HMR)
- RNase-free Water
- Depletion Master Mix components:
- Depletion Buffer
- RNase H
- DNase Digestion Master Mix components
- Fragment, Prime, and Elute Buffer (2X)
- RNase-free Water
- PEG/NaCl solution
- KAPA HiFi HotStart ReadyMix (2x)
- KAPA Library Amplification Primer Mix (10x)
Troubleshooting
Before start
Before starting protocol, ensure that the KAPA Pure beads are at room temperature. Also, prepare fresh 80% ethanol in molecular biology grade water.
RNA Sample Preparation
Concentrating RNA for library preparation is NOT necessary for all samples.
If concentration is needed, thaw 50 µl library aliquots of RNA samples on ice.
Poke hole in top of tube with 18G needle.
Concentrate in SpeedVac for ~40 min. Without heat. The volume will be ~12 µl.
After removing 10 µl concentrated RNA for library construction, place tape over hole in lid of original tube, and store remainder of RNA at -80°C.
Oligo Hybridization and RNA Depletion
Assemble RNA Hybridization master mix, keeping mixture at room temperature prior to use:
Note: All reagents are supplied with the kit, excluding the Custom DNA Oligo mix and the RNase-free water.
Add 10 µL master mix to each 0.2 ml tube.
Add 5 µL of RNase-free Water each tube.
Add 5 µL of RNA to each tube. Mix by pipetting up and down several times.
Place samples in the thermocycler and run hyb1 program:
During the ramp down to 45°C, prepare the depletion master mix and keep this mixture at room temperature prior to use:
When the program reaches 45°C, PAUSE the program and add 5 µL of the depletion master mix to each hybridization reaction and mix thoroughly by pipetting up and down several times.
Resume the cycling program to complete the depletion step.
Proceed immediately to rRNA Depletion Cleanup.
rRNA Depletion Cleanup
Add 55 µL KAPA Pure beads (2.2X volume) to each sample and pipette up and down at least 10 times to thoroughly mix.
Note: Incubate beads at room temperature for 15 minutes prior to use and vortex well.
Incubate the tube(s) at room temperature for 5 min to bind RNA to the beads.
Place the tube(s) on a magnet to capture the beads. Incubate until the liquid is clear (at least 2 min).
Carefully remove and discard supernatant.
Quick spin and remove and discard any trace amount of supernatant that remains.
Keep the tube(s) on the magnet, added 200 µL of 80% EtOH and incubate for 30 sec.
Carefully remove and discard the ethanol without disturbing the beads.
Quick spin the tube(s) and remove any residual ethanol without disturbing the beads.
DRY the beads at room temperature for 3 – 5 mins.
DNase Digestion
Assemble DNase Digestion master mix as follows, keeping this mixture at room temperature prior to use:
Thoroughly resuspend the beads in 22 µL of the DNase Digestion master mix by pipetting up and down multiple times.
Incubate the tube(s) at room temperature for 3 min to elute the RNA off the beads.
Place the tube(s) on a magnet and wait 1-2 min for the liquid is clear.
Carefully transfer 20 µL of supernatant into a tube(s). Discard the tube(s) with beads.
Place samples in the thermocycler and run dnase digestion program:
When the program reaches 4°C, proceed immediately to DNase Digestion Cleanup.
DNase Digestion Cleanup
Add 44 µL KAPA Pure beads (2.2X volume) to each sample and pipette up and down at least 10 times to thoroughly mix.
Note: Incubate beads at room temperature for 15 minutes prior to use and vortex well.
Incubate the tube(s) at room temperature for 5 min to bind RNA to the beads.
Place the tube(s) on a magnet to capture the beads. Incubate until the liquid is clear (at least 2 min).
Carefully remove and discard supernatant.
Quick spin and remove and discard any trace amount of supernatant that remains.
Keep the tube(s) on the magnet, added 200 µL of 80% EtOH and incubate for 30 sec.
Carefully remove and discard the ethanol without disturbing the beads.
Quick spin the tube(s) and remove any residual ethanol without disturbing the beads.
DRY the beads at room temperature for 3 – 5 mins.
RNA Elution, Fragmentation, and Priming
Prepare the required volume of Fragment, Prime and Elute Buffer (1X) by combining the following at room temperature:
Thoroughly resuspend the beads in 22 µL of the Fragment, Prime, and Elute buffer master mix by pipetting up and down multiple times.
Incubate the tube(s) at room temperature for 3 min to elute the RNA off the beads.
Place the tube(s) on a magnet to capture the beads. Incubate until the liquid is clear (at least 2 min).
Carefully transfer 20 µL of supernatant into a tube(s). Discard the tube(s) with beads.
Place samples in the thermocycler and run frag program.
When the program reaches 4°C, place tube(s) on ICE and proceed immediately to 1st Strand Synthesis.
1st Strand Synthesis
One ice, assemble the 1st strand synthesis master mix as follows:
Keeping the tube(s) on ice, add 10 µL of the 1st strand synthesis master mix to each tube and mix by pipetting up and down 10 times.
Place samples in the thermocycler and run 1st strand program:
When the program reaches 4°C, Place tube(s) on ICE and proceed immediately to 2nd Strand Synthesis and A-tailing.
2nd Strand Synthesis and A-Tailing
On ice, assemble the 2nd strand synthesis and A-tailing reaction master mix as follows:
Keeping the tube(s) on ice, add 30 µL of the 2nd strand synthesis and A-tailing reaction master mix to each tube and mix by pipetting up and down 10 times.
Place samples in the thermocycler and run 2nd strand program.
When the program reaches 4°C, place tube(s) on ICE and proceed immediately to Adapter Ligation.
Adapter Ligation
On ice, make up the ligation master mix as follow:
Add 5 µL of 1.5 uM adapter to each tube and mix by gently pipetting the mixture up and down multiple times.
Add 45 µL of Adapter Ligation Master Mix to each tube and pipette up and down 10 times to mix.
Place samples in the thermocycler and run adapter ligation program.
Proceed immediately to 1st Post-ligation Cleanup.
1st Post-Ligation Cleanup
In the same tube(s), add 70 µL KAPA Pure beads (0.63X volume) to each sample pipette up and down at least 10 times to thoroughly mix.
Note: Incubate beads at room temperature for 15 minutes prior to use and vortex well.
Incubate the tube(s) at room temperature for 5 min to bind DNA to the beads.
Place the tube(s) on a magnet to capture the beads. Incubate until the liquid is clear (at least 2 min).
Carefully remove and discard supernatant.
Quick spin and remove and discard any trace amount of supernatant that remains.
Keep the tube(s) on the magnet, added 200 µL of 80% EtOH and incubate for 30 sec.
Carefully remove and discard the ethanol without disturbing the beads.
Keep the tube(s) on the magnet, added 200 µL of 80% EtOH and incubate for 30 sec.
Carefully remove and discard the ethanol without disturbing the beads.
Quick spin the tube(s) and remove any residual ethanol without disturbing the beads.
DRY the beads at room temperature for 3 – 5 mins.
Remove the tube(s) from the magnet and thoroughly resuspend the beads in 50 µL of 10 mM Tris-HCl (pH 8.0 – 8.5).
Incubate the tube(s) at room temperature for 2 min to elute DNA of the beads.
Safe stopping point: The solution with resuspended beads can be stored at 4°C for ≤ 24 hrs. Do not freeze the beads. If stopping at this point, incubate samples at room temperature 15 min prior to use.
When ready, proceed to 2nd Post-ligation Cleanup.
2nd Post-Ligation Cleanup
In the same tube(s), add 35 µL PEG/NaCl solution (0.7X volume) to each sample and pipette up and down at least 10 times to thoroughly mix.
Note: PEG/NaCl solution should be room temperature to use.
Incubate the tube(s) at room temperature for 5 min to bind DNA to the beads.
Place the tube(s) on a magnet to capture the beads. Incubate until the liquid is clear (at least 2 min).
Carefully remove and discard supernatant.
Quick spin and remove and discard any trace amount of supernatant that remains.
Keep the tube(s) on the magnet, added 200 µL of 80% EtOH and incubate for 30 sec.
Carefully remove and discard the ethanol without disturbing the beads.
Keep the tube(s) on the magnet, added 200 µL of 80% EtOH and incubate for 30 sec.
Carefully remove and discard the ethanol without disturbing the beads.
Quick spin the tube(s) and remove any residual ethanol without disturbing the beads.
DRY the beads at room temperature for 3 – 5 mins.
Remove the tube(s) from the magnet and thoroughly resuspend the beads in 22 µL of 10 mM Tris-HCl (pH 8.0 – 8.5).
Incubate the tube(s) at room temperature for 2 min to elute DNA off the beads.
Place the tube(s) on a magnet to capture the beads and wait 1-2 minutes for the liquid to clear.
Transfer 20 µL of the supernatant to a new tube(s) and proceed to Library Amplification.
Safe stopping point: Sample can be stored at 4°C for 1 week, or at -20°C for 1 month.
Library Amplification
Assemble the library amplification master mix as follows:
Add 30 µL of the library amplification master mix to the 20 µL of purified, adapter-ligated DNA and mix by gently pipetting the mixture up and down 10 times.
Place samples in the thermocycler and run library amp program.
When the program reaches 4°C, proceed to Library Amplification Cleanup.
Library Amplification Cleanup
In the same tube(s), add 35 µL KAPA Pure beads (0.7X volume) to each sample and pipette up and down at least 10 times to thoroughly mix.
Note: Incubate beads at room temperature for 15 minutes prior to use and vortex well.
Incubate the tube(s) at room temperature for 5 min to bind DNA to the beads.
Place the tube(s) on a magnet to capture the beads. Incubate until the liquid is clear (at least 2 min).
Carefully remove and discard supernatant.
Quick spin and remove and discard any trace amount of supernatant that remains.
Keep the tube(s) on the magnet, added 200 µL of 80% EtOH and incubate for 30 sec.
Carefully remove and discard the ethanol without disturbing the beads.
Keep the tube(s) on the magnet, added 200 µL of 80% EtOH and incubate for 30 sec.
Carefully remove and discard the ethanol without disturbing the beads.
Quick spin the tube(s) and remove any residual ethanol without disturbing the beads.
DRY the beads at room temperature for 3 – 5 mins.
Remove the tube(s) from the magnet and thoroughly resuspend the beads in 22 µL of 10 mM Tris-HCl (pH 8.0 – 8.5).
Incubate the tube(s) at room temperature for 2 min to elute DNA off the beads.
Place the tube(s) on a magnet to capture the beads and wait 1-2 minutes for the liquid to clear.
Transfer 20 uL of the supernatant to a new tube(s) and store the purified, amplified libraries.
Quality Control
Measure DNA concentration using Qubit HS DNA assay.
Run libraries on an Agilent DNA 12000 bioanalyzer chip to determine final library size distribution and confirm minimal primer or adapter dimer carryover.