TITLE: Nano-OTS
AUTHORS: Ida Hoijer, Josefin Johansson, Sanna Gudmundsson, Chen-Shan Chin, Ignas Bunikis, Susana Häggkvist, Anastasia Emmanouilidou, Maria Wilbe,  Marcel Den Hoed,  Marie-Louise Bondeson,  Lars Feuk,  Ulf Gyllensten,  Adam Ameur

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This is a protocol that detects Cas9 off-target sites </span><span style = "font-style:italic;">in vitro</span><span> using nanopore sequencing</span><span style = "vertical-align:super;">1</span><span>. The protocol is a modified version published by Gilpatrick et al (2020)</span><span style = "vertical-align:super;">2</span><span>. Guide RNAs have been designed using CHOPCHOP (</span><a href="https://chopchop.cbu.uib.no" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://chopchop.cbu.uib.no/</span></a><span>).</span></div><div class = "text-block">1. Höijer, I., et al. (2020). "Amplification-free long read sequencing reveals unforeseen CRISPR-Cas9 off-target activity." bioRxiv: 2020.2002.2009.940486.</div><div class = "text-block"><span>2. Gilpatrick, T., Lee, I., Graham, J.E.</span><span style = "font-style:italic;">et al.</span><span>Targeted nanopore sequencing with Cas9-guided adapter ligation.</span><span style = "font-style:italic;">Nat Biotechnol</span><span style = "font-weight:bold;">38,</span><span>433–438 (2020). https://doi.org/10.1038/s41587-020-0407-5</span></div></div>

[STEPS]
?. [Shearing of genomic DNA]
Shear 5-10 µg of HMW genomic DNA to 20 kb using Megaruptor 2.
?. [Shearing of genomic DNA]
Add 0.45x AMPure XP beads to the sample and incubate at 2000 rpm using a vortex mixer for 10 min.
?. [Shearing of genomic DNA]
Place the tube on a magnetic rack until the beads collect on the side of the tube and the solution appears to be clear. Slowly pipette off the supernatant and discard. Be careful not to disturb the beads.
?. [Shearing of genomic DNA]
Wash the beads using 70% freshly prepared ethanol.     - Fill the tube with the ethanol    - Do not disurb the bead pellet    - Wait 30 seconds and pipette off the ethanol and discard
?. [Shearing of genomic DNA]
Repeat step 2.2
?. [Shearing of genomic DNA]
Remove any residual ethanol.    - Spin the tube briefly and put back on the magnetic rack    - Pipette off any remaining ethanol using a P10 pipette
?. [Shearing of genomic DNA]
Add 32 µl Low TE buffer and incubate at 2000 rpm using a vortex mixer for 5 min to elute the sample.
?. [Shearing of genomic DNA]
Place the tube on the magnetic rack until the beads collect on the side of the tube and the solution appears clear. Slowly pipette off the supernatant and transfer to a new eppendorf tube.
?. [Quality control of fragmented DNA]
Measure concentration of the sample using Qubit and fragment size using Bioanalyzer, Tape station or FEMTO Pulse.
?. [Size selection of sheared DNA]
Perform size selection with a 10 kb cut-off using the BluePippin and the High pass plus gel casette with the "15 kb High Pass Plus Marker U1" casette definition (the 10 kb cut-off is not supported by Sage Science).
?. [Size selection of sheared DNA]
Add 0.45x AMPure XP beads to the sample and incubate at 2000 rpm using a vortex mixer for 10 min.
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?. [Size selection of sheared DNA]
Place the tube on a magnetic rack until the beads collect on the side of the tube and the solution appears to be clear. Slowly pipette off the supernatant and discard. Be careful not to disturb the beads.
?. [Size selection of sheared DNA]
Wash the beads using 70% freshly prepared ethanol.     - Fill the tube with the ethanol    - Do not disurb the bead pellet    - Wait 30 seconds and pipette off the ethanol and discard
?. [Size selection of sheared DNA]
Repeat step 5.2
?. [Size selection of sheared DNA]
Remove any residual ethanol.     - Spin the tube briefly and put back on the magnetic rack    - Pipette off any remaining ethanol using a P10 pipette
?. [Size selection of sheared DNA]
Add 24 µl Low TE buffer and incubate at 2000 rpm using a vortex mixer for 5 min to elute the sample.
?. [Size selection of sheared DNA]
Place the tube on the magnetic rack until the beads collect on the side of the tube and the solution appears clear. Slowly pipette off the supernatant and transfer to a new eppendorf tube.
?. [Preparing the Cas9 ribonucleoprotein complexes (RNPs)]
Resuspend crRNAs  and tracrRNA to a final concentration of 100 µM in TE pH 7.5.
?. [Preparing the Cas9 ribonucleoprotein complexes (RNPs)]
Pool all crRNAs used in the same experiment equimolarly by combining 1 µl of each crRNA in a new eppendorf tube. Unused crRNa mix can be stored at -70ºC.
?. [Preparing the Cas9 ribonucleoprotein complexes (RNPs)]
Anneal crRNA and tracrRNA. In a 0.2 ml tube (preferably low-bind) combine:        8 µl Duplex buffer        1 µl crRNA pool (100 µM)        1 µl tracrRNA (100 µM)Inclubate at 95ºC for 5 min in a thermocycler. Remove the tube from the thermocycler and let it cool down to room temperature (~5 min). Place the tube on ice.
?. [Preparing the Cas9 ribonucleoprotein complexes (RNPs)]
Assemble the RNPs. Add the following components to a new 1.5 ml eppendorf tube:        5 µl annealed crRNA-tracrRNA        5 µl 10x NEB CutSmart buffer        39.6 µl nuclease-free water        0.4 µl HiFi Cas9 (62 µM)Incubate at room temperature for 30 min, then place on ice.While the RNPs are incubating, proceed to the next step.
?. [Dephosphorylation of sheared and size selected DNA]
3 µg of sheared and size selected DNA is needed for this step. Bring the sample to 24 µl using nuclease-free water if needed.
?. [Dephosphorylation of sheared and size selected DNA]
Add the following components in a 0.2 ml thin-walled PCR tube:        3 µl 10x NEB Cut Smart buffer        24 µl sheared and size selected DNA (3 µg)Mix gently by flicking the tube, spin down.Add 3 µl CIP to the tube.Incubate at 37ºC for 10 min, 80ºC for 2 min and then hold at 20ºC on a thermocycler.
?. [Cas9 digestion and a-tailing]
Make a 10 mM dATP solution by combining 1 µl 100mM ATP and 9 µl nuclease-free water. Do not keep the solution for future experiments.
?. [Cas9 digestion and a-tailing]
Make a 1x NEB Cut Smart solution by combining 1 µl 10x NEB Cut Smart Buffer and 9 µl nuclease-free water. Do not keep the solution for future experiments.
?. [Cas9 digestion and a-tailing]
Add the following components to the PCR tube containing the dephosphorylated DNA sample:        30 µl dephosyphorylated DNA        7 µl 1x NEB Cut smart buffer        3 µl RNPs        1 µl 10 mM dATP        1 µl NEB Taq polymeraseMix carefully by inverting the tube, spin down.Incubate at 37ºC for 15 min, 72ºC for 5 min and hold at 4ºC. Then place the tube on ice.
?. [Adapter ligation]
Carefully transfer the contents of the 0.2 ml PCR-tube to a new 1.5 ml eppendorf tube.
?. [Adapter ligation]
Add the following compontents to a separate 1.5 ml eppendorf tube. It is important to add the adapter mix (AMX) last.        20 µl ligation buffer (LNB)        3 µl nuclease-free water        10 µl NEBNext Quick T4 DNA ligase        5 µl adapter mix (AMX)Mix thoroughly  by pipetting.
?. [Adapter ligation]
Add 20 µl of the adapter ligation mix to the Cas9 digested and dA-tailed DNA sample. Mix gently by flicking the tube. Do not spin down at this stage.
?. [Adapter ligation]
Immediately after mixing, add the remaining adapter ligation mix. Mix gently by flicking the tube, spin down.Inclubate at room temperature for 10 min.
?. [AMPure XP bead purification]
Add 1 volume (80 µl) of TE pH 8.0 to the adapter ligated sample. Mix gently by flicking the tube.
?. [AMPure XP bead purification]
Add 0.3x (48 µl) AMPure XP beads to the sample. Mix by gentle inversion.Incubate at room temperature for 10 min. Do not agitate the sample.
?. [AMPure XP bead purification]
Spin down the sample and place on a magnetic rack until the beads collect on the side of the tube and the solution appears to be clear. Pipette off the supernatant and discard. Be careful not to disturb the pellet.
?. [AMPure XP bead purification]
Wash the beads by adding 250 µl long fragment buffer (LFB). Resuspend the beads by flicking the tube and place back on the magnetic rack.When the solution appears clear, pipette off the supernatant and discard.
?. [AMPure XP bead purification]
Repeat step 22.
?. [AMPure XP bead purification]
Remove any residual buffer.     - Spin the tube briefly and put back on the magnetic rack    - Pipette off any remaining buffer using a P10 pipette
?. [AMPure XP bead purification]
Remove the tube from the magnetic rack and add 13 µl elution buffer (EB) to elute the sample. Incubate at room temperature for 10 min. Do not agitate the sample.
?. [AMPure XP bead purification]
Place the tube on the magnetic rack until the beads collect of the side of the tube and the solution appears clear. Pipette off 12 µl of the supernantant and transfer to a new 1.5 ml eppendorf tube. Place on ice.
?. [Sequencing on the MinION]
Open the priming port on the MinION flow cell. Draw back a small volume to remove any bubbles by:- Set a P1000 to 200 µl- Insert the tip into the priming port- Turn the wheel of the pipette until the dial shows 220-230 µl, or until you can see a small volume of buffer in the pipette tip
?. [Sequencing on the MinION]
Add 30 µl of flush tether (FLT) directly to the flush buffer (FLB). Mix by pipetting. Load 800 µl of the priming mix to the priming port. Avoid the introduction of bubbles. Wait 5 min.
?. [Sequencing on the MinION]
Add the following components to a new 1.5 ml eppendorf tube:        25 µl sequencing buffer (SQB)        13 µl loading beads (LB)        12 µl DNA library
?. [Sequencing on the MinION]
Complete the flow cell binding by:- Gently open the SponON sample port cover- Load 200 µl of the priming mix to the priming port. Avoid the introdution of bubbles.
?. [Sequencing on the MinION]
Mix the prepared library gently by pipetting just prior to loading.Add 50 µl of the library SpotON sample port in a dropwise fashion. Ensure each drop flows into the port before adding the next one.
?. [Sequencing on the MinION]
Start the sequncing run through the MinKNOW software.
?. [Data analysis]
Align the reads using minimap2. Use the mapped reads for detection of off-target sites using our custom tool Insider:https://github.com/UppsalaGenomeCenter/InSiDeR