Jul 09, 2025

Public workspaceDASH Protocol v2.5

 Forked from DASH Protocol v2.5
DOI
https://dx.doi.org/10.17504/protocols.io.n2bvjerbwgk5/v1
  • Amy Lyden1,
  • Emily Crawford1,
  • Jenai Quan1,
  • Saharai Caldera2,
  • David Dynerman1,
  • Karel Foucault3
  • 1CZ Biohub;
  • 2UCSF;
  • 3Google
  • Amy Lyden: Zeus;
Karel Foucault
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DOI: https://dx.doi.org/10.17504/protocols.io.n2bvjerbwgk5/v1
Protocol Citation: Amy Lyden, Emily Crawford, Jenai Quan, Saharai Caldera, David Dynerman, Karel Foucault 2025. DASH Protocol v2.5. protocols.io https://dx.doi.org/10.17504/protocols.io.n2bvjerbwgk5/v1
Manuscript citation:

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: July 09, 2025
Last Modified: July 09, 2025
Protocol Integer ID: 222139
Keywords: CRISPR, sequencing, cas9, depletion, ribosomal, useful for rna, guide rna, sequencing library, human mitochondrial ribosomal rna, seq of human metagenomic sample, set of guide rna, unique barcoding of the rna, rna, human metagenomic sample, abundant species such as human mitochondrial ribosomal rna, dash treatment, crispr, dash protocol, rrna, sequencing space, ligation of adapter, dash, ligation, seq library, hybridization
Funders Acknowledgements:
Karel
Grant ID: 007king
Abstract
This protocol is for performing Depletion of Abundant Sequences by Hybridization (DASH) after preparing sequencing libraries and pooling together.

DASH is most useful for RNA-seq of human metagenomic samples, where abundant species such as human mitochondrial ribosomal RNAs (rRNAs) occupy a majority of the sequencing space available, leaving a minor fraction for regions of interest.
DASH treatment is performed after ligation of adapters and unique barcoding of the RNA-seq library. It employs CRISPR-Cas9 complexed to a set of guide RNAs (gRNAs) targeted to the abundant regions to be depleted in a given library. These abundant regions in the library are then cleaved, leaving only the fragments with intact adapters on both ends to be further amplified and sequenced.



Guidelines
This protocol describes DASH for an RNA library prepared using the NEBNext Ultra II RNA Library Prep Kit. Other standard library preparation kits may be used instead, as long as DASH is applied after ligation of adapters.
Materials
MATERIALS
ReagentUSER Enzyme - 250 unitsNew England BiolabsCatalog #M5505L
ReagentNEBNext Adaptor for IlluminaNew England BiolabsCatalog #E7337 in Kits E7335, E7500, E771
ReagentThermocycler
ReagentNEBNext Ultra II RNA Library Prep Kit for Illumina - 96 rxnsNew England BiolabsCatalog #E7770L
ReagentNuclease-free water AmbionCatalog #AM9932
ReagentQubit dsDNA HS kit Life TechnologiesCatalog #Q32851
Reagent Bioanalyzer chips and reagents (DNA High Sensitivity kit)Agilent Technologies
ReagentTruSeq i7/i5 Indexing Primers - Custom (or NEBNext® Multiplex Oligos for Illumina)New England BiolabsCatalog #E7500L
ReagentCas9 40µM
ReagentDual guide RNAs (40µM - targeted to genes or regions to be depleted - crisprRNA and tracr RNA - quantified by RNA Qubit)
Reagent10X Cas9 Activity Buffer (500nM Tris pH 8.0 100nM MgCl2 10nM TCEP)
ReagentProteinase KNew England BiolabsCatalog #P8107S
ReagentSPRI beads (homemade) or Ampure XP beads
ReagentKapa HiFi Real-Time Amplification KitKapa BiosystemsCatalog #KK2702
ReagentZymo DNA Clean & Concentrator - 5Zymo ResearchCatalog #D4014
ReagentMagnetic rack for PCR strips
ReagentqPCR machine
STEP MATERIALS
ReagentCas9 40µM
Reagent10X Cas9 Activity Buffer (500nM Tris pH 8.0 100nM MgCl2 10nM TCEP)
ReagentDual guide RNAs (40µM - targeted to genes or regions to be depleted - crisprRNA and tracr RNA - quantified by RNA Qubit)
ReagentZymo DNA Clean & Concentrator - 5Zymo ResearchCatalog #D4014
ReagentProteinase KNew England BiolabsCatalog #P8107S
ReagentSPRI beads (homemade) or Ampure XP beads
Reagent Bioanalyzer chips and reagents (DNA High Sensitivity kit)Agilent Technologies
ReagentQubit dsDNA HS kit Life TechnologiesCatalog #Q32851
ReagentKapa HiFi Real-Time Amplification KitKapa BiosystemsCatalog #KK2702
ReagentSPRI beads (homemade) or Ampure XP beads
ReagentQubit dsDNA HS Assay kit Thermo Fisher ScientificCatalog #Q32854
Reagent Bioanalyzer chips and reagents (DNA High Sensitivity kit)Agilent Technologies
ReagentQubit dsDNA HS Assay KitThermo Fisher ScientificCatalog #Q32851
Reagent Bioanalyzer chips and reagents (DNA High Sensitivity kit)Agilent Technologies
ReagentNEBNext Ultra II RNA Library Prep Kit for Illumina - 96 rxnsNew England BiolabsCatalog #E7770L

Protocol materials
ReagentNEBNext Ultra II RNA Library Prep Kit for Illumina - 96 rxnsNew England BiolabsCatalog #E7770L
ReagentQubit dsDNA HS Assay KitThermo Fisher ScientificCatalog #Q32851
Reagent Bioanalyzer chips and reagents (DNA High Sensitivity kit)Agilent Technologies
ReagentDual guide RNAs (40µM - targeted to genes or regions to be depleted - crisprRNA and tracr RNA - quantified by RNA Qubit)
ReagentCas9 40µM
Reagent10X Cas9 Activity Buffer (500nM Tris pH 8.0 100nM MgCl2 10nM TCEP)
ReagentZymo DNA Clean & Concentrator - 5Zymo ResearchCatalog #D4014
ReagentProteinase KNew England BiolabsCatalog #P8107S
ReagentSPRI beads (homemade) or Ampure XP beads
ReagentQubit dsDNA HS kit Life TechnologiesCatalog #Q32851
ReagentKapa HiFi Real-Time Amplification KitKapa BiosystemsCatalog #KK2702
ReagentQubit dsDNA HS Assay kit Thermo Fisher ScientificCatalog #Q32854
Troubleshooting
Before start
Prepare your sequencing libraries, and make sure you have gRNAs for DASH. We used the DASHit software to generate an optimized set of guide RNA sequences based on a dataset of unwanted genes. (For DASHit software, visit: https://github.com/czbiohub/guide_design_tools/blob/master/dashit/dashit-reads/dashit-reads.org). You can buy the gRNAs or buy DNA templates and transcribe them. For an IVT protocol for crispr RNA and tracr RNA, please contact [email protected] or [email protected])
Prepare indexed RNA-seq library
Follow all steps using NEB Ultra II RNA library preparation kit. Use an input RNA volume of 25ng if available, or less if not available. Perform 12-18 cycles of indexing PCR.
ReagentNEBNext Ultra II RNA Library Prep Kit for Illumina - 96 rxnsNew England BiolabsCatalog #E7770L











Choose one option:

a. Pooled DASH: If there are multiple samples, you may pool them at normalized concentrations of DNA between 200 – 1000 bp by Qubit or BioA/Tapestation/Fragment Analyzer, or using a preliminary low-depth sequencing run (such as an iSeq or MiSeq) of an equivolume pool to determine pooling ratios. Alternatively, if you suspect that your samples may vary drastically in DASHable material, you can pool equivolumes of your samples and DASH, then run an iSeq or MiSeq sequencing. Then pool normalized concentrations based on these values. This pooled library will go into a single DASH reaction.

b. Single Sample DASH: Additionally if you suspect your samples may vary in DASHable material or have a high amount of DASHable material, you may DASH each sample individually and pool them after. In this case, each sample will have its own DASH reaction, and the following steps will be performed on each individual library.
Quantify by HS DNA Qubit.
ReagentQubit dsDNA HS Assay KitThermo Fisher ScientificCatalog #Q32851

Perform a HS DNA Bioanalyzer (or other fragment analysis) and ensure for your library(ies):

1) that adapter dimers have been removed. Several SPRI bead clean-ups at a sample:bead volume ratio of 1:1 may be necessary.
2) there is sufficient sample in between the 200 – 1000 bp range.
3) There is no ‘PCR bubble’ (appears as secondary bump in fragment analysis trace at higher bp regions, see trace below). PCR bubble will be eliminated with 1-2 extra PCR cycles. We recommend.
  • 100ng of DNA in 23µL H20
  • 25µL Kapa HiFi Real-Time Amplification Master Mix
  • 2µL Illumina primers at 5µM.
Library pre-PCR cycles with DNA in 2000-10000bp region. This library may be over-amplified and cannot be used in DASH.
Library post 2xPCR cycles with no DNA in 2000-10000bp region. This confirms library previously had PCR bubble. This library can be used in DASH.


Reagent Bioanalyzer chips and reagents (DNA High Sensitivity kit)Agilent Technologies

Each DASH reaction requires at least 10 µL of 2.8 nM DNA library (approximately 0.83 ng/µL for an average 450 bp library). You may concentrate your sample by SPRI bead clean-up if necessary.
DASH
Thaw necessary reagents and let come to room temperature before use. If you will not be using for more than 10 minutes, put on ice and take off 5 minutes prior to mixing.
ReagentCas9 40µM

Reagent10X Cas9 Activity Buffer (500nM Tris pH 8.0 100nM MgCl2 10nM TCEP)


ReagentDual guide RNAs (40µM - targeted to genes or regions to be depleted - crisprRNA and tracr RNA - quantified by RNA Qubit)

Prepare master mix (MM) of Cas9 and gRNAs:

Note
Ensure that the dual-guide RNAs have been annealed at 95°C for 30 seconds and then cooled to room temperature. No additional heating is recommended if pairs were annealed prior to storage.

Note
Cas9-gRNAs MM will be replenished once in the protocol. Ensure you have enough reagents for 2X MM for each sample.

ReagentStock ConcentrationFinal Concentration1X cas9-gRNA MM
10X cas9 buffer10X1.25X2.5µL
cas940µM5µM2.5µL
dual-guide RNAs40µM10µM5µL
10µL total
DASH Master Mix

Mix well by gently pipetting and tapping and incubate this mixture at Temperature37 °C forDuration00:05:00 .


Add Amount10 µL of a 2.8 nM barcoded DNA library (pooled or not) to every Amount10 µL of the above Cas9-gRNA mix.
o This reaction may be scaled up from the original 1X (Amount20 µL DASH reaction)
o Calculator for µg moles: https://www.promega.com/a/apps/biomath/


ReagentStock ConcentrationFinal Concentration1X cas9-gRNA MM
10X cas9 buffer10X1.25X2.5µL
cas940µM5µM2.5µL
dual-guide RNAs40µM10µM5µL
DNA library2.8nM1.4nM10µL
20µL total
Final DASH reaction (1X, 20µL)



Incubate the DASH reaction at Temperature37 °C for Duration00:30:00 .


Before your incubation is up, prepare a second batch of 1X Cas9-gRNA MM for all your samples and incubate mixture at Temperature37 °C for Duration00:05:00 .


After incubation, perform a column clean up. Use the Zymo DNA Clean & Concentrate - 5 kit to purify your reaction. Follow the kit instructions for PCR product (Amount100 µL of binding buffer to Amount20 µL of DNA in DASH reaction). Elute inAmount10.5 µL H20, and then reload all Amount10.5 µL onto the column, and elute again.

ReagentZymo DNA Clean & Concentrator - 5Zymo ResearchCatalog #D4014





Add Amount10 µL of your eluted DNA to Amount10 µL of 1X Cas9-gRNA MM and mix by pipette.


Incubate the DASH reaction atTemperature37 °C for Duration01:30:00 .
Note
Total incubation of DNA library in Cas9-gRNA MM should be Duration02:00:00 . Column clean-up timing has not been optimized, and could be split to beTemperature37 °C for Duration01:00:00 , column clean, and then Temperature37 °C for Duration01:00:00 .



Add Amount1 µL proteinase K to each Amount20 µL reaction and mix well by gently tapping or pipetting.

ReagentProteinase KNew England BiolabsCatalog #P8107S



Incubate at Temperature50 °C for Duration00:15:00 for cas9 inactivation.


Clean up DASH reaction
Prepare to do a SPRI selection of sample:bead 1:0.9 (equivalent to Ampure 0.7X)
Note
This goal of this step is to purify your target library away from the DASH reagents (buffers, Cas9, gRNAs) and the DASH-digested fragments. Depending on DASH efficiency and the size of the expected digested fragments, this ratio may be altered to best remove the fragments.

Note
Other magnetic beads such as AmpureXP may be used instead of SPRI. However, please take note of the different size cut-offs for sample:bead ratios, as they may vary from the homebrew SPRI beads used in this protocol.

Equilibrate SPRI beads to room temperature and vortex well to mix.

ReagentSPRI beads (homemade) or Ampure XP beads

Add beads equivalent to 0.9X the sample volume to each sample tube (for Amount21 µL of sample, add Amount18.9 µL beads).


Mix well by pipetting or tapping the tubes and spin down briefly.
Incubate for Duration00:05:00 at room temperature, then put the tubes on the magnetic rack. Allow beads to separate on the magnet for 3-5 minutes, or until the supernatant is clear.

Keeping the tubes on the magnet, carefully remove and discard the supernatant.
Add Amount200 µL 80% ethanol (prepared fresh). Incubate beads for Duration00:01:00 and then remove the ethanol.


Repeat the above ethanol wash step.
Allow the beads to air dry for approximately Duration00:05:00 . Do not over-dry. Dry beads should appear matte (rather than glossy) but should not have a cracked appearance. Overdried beads may not resuspend or elute well.

Remove tubes from magnet and addAmount27 µL nuclease-free H2O.

Resuspend well by vortexing, tapping the tubes, and spinning down briefly.
Allow Duration00:02:00 for DNA to elute from beads, then transfer tubes back to magnet

Allow the beads to separate on magnet.
CollectAmount25 µL of supernatant from each sample and transfer it to a clean PCR tube.

Run a fragment analysis, such as with the HS dsDNA Bioanalyzer kit or the Agilent HS D5000 Tapestation kit.

Reagent Bioanalyzer chips and reagents (DNA High Sensitivity kit)Agilent Technologies



Expected result
If DASH is successful, the trace may be characterized by the presence of small peaks between 30 – 100 bp. The size of these peaks will depend on the proportion of expected depleted product over target product, and how closely spaced together the target guide RNAs were designed.

DASHed sample showing the digested fragments and gRNAs (30-140), an adapter dimer (178), and low concentration of target library (sloping from 300 – 1000+). Bioanalyzer internal markers in green (35) and purple (10380). This sample needs additional cleanup and amplification.

If desired, also quantify by HS DNA Qubit.

ReagentQubit dsDNA HS kit Life TechnologiesCatalog #Q32851

As needed, perform additional clean-up steps to remove the digested fragments and adapter dimers in your sample. Proceed to amplification of target library if your concentration in the 200-1000bp region is very low, or looks nonexistent.
Amplification of Target Library
Perform this Real-Time PCR amplification step if after cleaning out the digested fragments and adapter dimers your library concentration is too low for loading onto a sequencer.

Note
This step could be optional, if your DASHable material is low and your concentration after DASH is high. However, we have found it to be almost always necessary.

Thaw Kapa HiFi Real-Time Amplification Master Mix and Standard 2 on ice

ReagentKapa HiFi Real-Time Amplification KitKapa BiosystemsCatalog #KK2702

Prepare the following reaction for each library:

ReagentVolume (1X)
2X Kapa HiFi Real-time Amplification Master Mix25µL
5µM Universal Illumina Primers (5sol-20&21)2µL
DNA (bring up volume with H20 if needed)23µL
50µL

Place each sample in a tube or strip of tubes that is physically separated from the others, so that they can be removed one at a time.
Put Amount50 µL of Standard 2 in a PCR tube

Cycle with the following conditions in an RT-PCR machine. Make sure that if your machine does a baseline correction, that you remove it.

TemperatureTimeCycles
98C45 sec1
98C15 sec25
63C30 sec
72C1m 45 sec
Plate read
72C
Cycling conditions for PCR


Watch the qPCR graph during the plate read. When a sample reaches the fluorescence threshold given by standard 2, remove that sample DURING the 72°C for 20 second timeframe after the plate read.
Perform a SPRI cleanup as described in `Clean up DASH reaction` section above at a sample:bead ratio of 1:0.9 to remove amplification reagents, and elute in a suitable volume of water. Go to

ReagentSPRI beads (homemade) or Ampure XP beads


Quantify and analyze your library by HS DNA Qubit and HS dsDNA Bioanalyzer.

ReagentQubit dsDNA HS Assay kit Thermo Fisher ScientificCatalog #Q32854

Reagent Bioanalyzer chips and reagents (DNA High Sensitivity kit)Agilent Technologies

Expected result
This trace should resemble a regular library with a majority of the library sized between 200 – 1000bp:


Same DASHed sample from above.SPRI cleaned at sample:bead ratio 1:0.7, 2 amplification cycles. Small primer dimer at 140bp.

Perform more SPRI clean-ups or amplification if necessary.
Submit your library for sequencing!