TITLE: DASH Protocol v2.5
AUTHORS: Amy Lyden, Emily Crawford, Jenai Quan, Saharai Caldera, David Dynerman

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is for performing Depletion of Abundant Sequences by Hybridization (DASH) after preparing sequencing libraries and pooling together.</div><div class = "text-block">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. </div><div class = "text-block">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.</div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/pr-journal/berna.png" /></div></div>

[STEPS]
?. [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.
?. [Prepare indexed RNA-seq library]
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.
?. [Prepare indexed RNA-seq library]
Quantify by HS DNA Qubit.
?. [Prepare indexed RNA-seq library]
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.
?. [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.
?. [DASH]
Prepare master mix (MM) of Cas9 and gRNAs:  ABCD1ReagentStock ConcentrationFinal Concentration1X cas9-gRNA MM210X cas9 buffer10X1.25X2.5µL3cas940µM5µM2.5µL4dual-guide RNAs40µM10µM5µL510µL total
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.
Cas9-gRNAs MM will be replenished once in the protocol. Ensure you have enough reagents for 2X MM for each sample.
ABCD1ReagentStock ConcentrationFinal Concentration1X cas9-gRNA MM210X cas9 buffer10X1.25X2.5µL3cas940µM5µM2.5µL4dual-guide RNAs40µM10µM5µL510µL total
?. [Prepare indexed RNA-seq library]
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]
Mix well by gently pipetting and tapping and incubate this mixture at for.
37 °C
?. [DASH]
Add  of a 2.8 nM barcoded DNA library (pooled or not) to every  of the above Cas9-gRNA mix. o This reaction may be scaled up from the original 1X ( DASH reaction)o Calculator for µg moles: https://www.promega.com/a/apps/biomath/  ABCD1ReagentStock ConcentrationFinal Concentration1X cas9-gRNA MM210X cas9 buffer10X1.25X2.5µL3cas940µM5µM2.5µL4dual-guide RNAs40µM10µM5µL5DNA library2.8nM1.4nM10µL620µL total
10 µl
10 µl
20 µl
ABCD1ReagentStock ConcentrationFinal Concentration1X cas9-gRNA MM210X cas9 buffer10X1.25X2.5µL3cas940µM5µM2.5µL4dual-guide RNAs40µM10µM5µL5DNA library2.8nM1.4nM10µL620µL total
?. [DASH]
Incubate the DASH reaction at for .
37 °C
?. [DASH]
Before your incubation is up, prepare a second batch of 1X Cas9-gRNA MM for all your samples and incubate mixture at  for .
37 °C
?. [DASH]
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 (of binding buffer to of DNA in DASH reaction). Elute inH20, and then reload all onto the column, and elute again.
100 µl
20 µl
10.5 µl
10.5 µl
?. [DASH]
Add of your eluted DNA to  of  1X Cas9-gRNA MM and mix by pipette.
10 µl
10 µl
?. [DASH]
Incubate the DASH reaction atfor .
37 °C
Total incubation of DNA library in Cas9-gRNA MM should be  . Column clean-up timing has not been optimized, and could be split to be  for  , column clean, and then   for  .
?. [DASH]
Add  proteinase K to each  reaction and mix well by gently tapping or pipetting.
1 µl
20 µl
?. [DASH]
Incubate at for for cas9 inactivation.
50 °C
?. [Clean up DASH reaction]
Prepare to do a SPRI selection of sample:bead 1:0.9 (equivalent to Ampure 0.7X)
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.
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.
?. [Clean up DASH reaction]
Equilibrate SPRI beads to room temperature and vortex well to mix.
?. [Clean up DASH reaction]
Add beads equivalent to 0.9X the sample volume to each sample tube (for  of sample, add  beads).
21 µl
18.9 µl
?. [Clean up DASH reaction]
Mix well by pipetting or tapping the tubes and spin down briefly.
?. [Clean up DASH reaction]
Incubate for  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.
?. [Clean up DASH reaction]
Keeping the tubes on the magnet, carefully remove and discard the supernatant.
?. [Clean up DASH reaction]
Add 80% ethanol (prepared fresh). Incubate beads for  and then remove the ethanol.
200 µl
?. [Clean up DASH reaction]
Repeat the above ethanol wash step.
?. [Clean up DASH reaction]
Allow the beads to air dry for approximately . 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.
?. [Clean up DASH reaction]
Remove tubes from magnet and add nuclease-free H2O.
27 µl
?. [Clean up DASH reaction]
Resuspend well by vortexing, tapping the tubes, and spinning down briefly.
?. [Clean up DASH reaction]
Allow  for DNA to elute from beads, then transfer tubes back to magnet
?. [Clean up DASH reaction]
Allow the beads to separate on magnet.
?. [Clean up DASH reaction]
Collectof supernatant from each sample and transfer it to a clean PCR tube.
25 µl
?. [Clean up DASH reaction]
Run a fragment analysis, such as with the HS dsDNA Bioanalyzer kit or the Agilent HS D5000 Tapestation kit.
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.
?. [Clean up DASH reaction]
If desired, also quantify by HS DNA Qubit.
?. [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.
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.
?. [Clean up DASH reaction]
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]
Thaw Kapa HiFi Real-Time Amplification Master Mix and Standard 2 on ice, plus Illumina P5 and P7 primers
?. [Amplification of Target Library]
Prepare the following reaction for each library:  AB1ReagentVolume (1X)22X Kapa HiFi Real-time Amplification Master Mix25µL35µM Illumina P5 and P7 primers2µL4DNA (bring up volume with H20 if needed)23µL550µL
AB1ReagentVolume (1X)22X Kapa HiFi Real-time Amplification Master Mix25µL35µM Illumina P5 and P7 primers2µL4DNA (bring up volume with H20 if needed)23µL550µL
?. [Amplification of Target Library]
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.
?. [Amplification of Target Library]
Put  of Standard 2 in a PCR tube
50 µl
?. [Amplification of Target Library]
Cycle with the following conditions in an RT-PCR machine. Make sure that if your machine does a baseline correction, that you remove it.  ABC1TemperatureTimeCycles298C45 sec1398C15 sec25463C30 sec572C1m 45 sec6Plate read772C
ABC1TemperatureTimeCycles298C45 sec1398C15 sec25463C30 sec572C1m 45 sec6Plate read772C
?. [Amplification of Target Library]
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.
?. [Amplification of Target Library]
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.
?. [Amplification of Target Library]
Quantify and analyze your library by HS DNA Qubit and HS dsDNA Bioanalyzer.
This trace should resemble a regular library with a majority of the library sized between 200 – 1000bp:
?. [Amplification of Target Library]
Perform more SPRI clean-ups or amplification if necessary.
?. [Amplification of Target Library]
Submit your library for sequencing!