Nov 15, 2024

Public workspaceCas9-targeted enrichment of MEIs for ONT R9 flow cells

This protocol is a draft, published without a DOI.
  • 1University of Michigan;
  • 2University of Michigan - Ann Arbor
Jessica Switzenberg
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Protocol Citation: Jessica Switzenberg, Camille Mumm, Torrin McDonald, Alan Boyle, Alan Boyle 2024. Cas9-targeted enrichment of MEIs for ONT R9 flow cells. Protocol exchange https://protocols.io/view/cas9-targeted-enrichment-of-meis-for-ont-r9-flow-c-drnc55aw
Manuscript citation:
McDonald, T.L., Zhou, W., Castro, C.P. et al. Cas9 targeted enrichment of mobile elements using nanopore sequencing. Nat Commun 12, 3586 (2021). https://doi.org/10.1038/s41467-021-23918-y
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 have used this protocol for ONT R9 chemistry and it works very well.
Created: November 13, 2024
Last Modified: November 15, 2024
Protocol Integer ID: 112036
Keywords: nCATS, Cas9, enrichment, MEI
Abstract
This is the Boyle lab's Cas9-targeted enrichment for mobile element insertions within genomic DNA isolated from cell lines. We use ONT R9 flow cells for long-read sequencing following Cas9 enrichment. Based on our published L1H enrichment, we expect 50x or greater coverage over the targeted MEI.
Materials
Nuclei Lysis Buffer solution:
10mM Tris-HCl, pH 8
400mM NaCl
2mM EDTA, pH 8

EnGen sgRNA Synthesis Kit (NEB, E3322S)
Molecular biology grade water
Qubit instrument w/dsDNA BR and HS assay kit; RNA BR assay kit
Alt-R S.p. Cas9 HiFi V3 nuclease (IDT)
10X Cutsmart Buffer (NEB, can also use rCutsmart Buffer without any issues)
Taq DNA polymerase (NEB, M0273L)
10mM dATP
T4 DNA ligase (NEB, M0202M)
SQK-LSK109 kit from Oxford Nanopore Technologies
MinION R9 flow cell
MinION sequencer

Protocol materials
ReagentRNase AQiagenCatalog #19101
Step 11
ReagentSodium Chloride, 5 M
Step 15
Reagent1X TE buffer (10 mM Tris-HCl pH 8.0 1 mM EDTA)
In 3 steps
Reagent3M Sodium Acetate solution
Step 39
ReagentChloroform
In 2 steps
ReagentSDS, 10% SolutionLife TechnologiesCatalog #AM9822
Step 10
ReagentProteinase KMerck MilliporeSigma (Sigma-Aldrich)Catalog #3115879001
Step 13
Reagent100% Ethanol
In 2 steps
ReagentTRIzol ReagentThermo Fisher ScientificCatalog #15596026
Step 31
High Molecular Weight (HMW) genomic DNA (gDNA) extraction from cell lines
High Molecular Weight (HMW) genomic DNA (gDNA) extraction from cell lines
1h 25m
1h 25m
Pellet cells in swinging bucket centrifuge atCentrifigation500 x g, 4°C Duration00:10:00

10m
Remove supernant
Wash cell pellets in cold Reagent1X PBS (Phosphate-buffered saline )

Go togo to step #1

10m
Add Amount5 mL of cold Reagent1X PBS (Phosphate-buffered saline ) to cell pellet and resuspend the cells

Count cells using your method of choice; we used a Countess FL
Move forward with the number of cells you need - We recommend at least 20M cells
Go togo to step #1

10m
Resuspend the cell pellet in Amount3 mL of nuclei lysis buffer

Add Amount200 µL of ReagentSDS, 10% SolutionLife TechnologiesCatalog #AM9822 to the resuspension and invert to mix evenly

Add Amount50 µL of ReagentRNase AQiagenCatalog #19101

Rotate for Duration00:30:00 at Temperature37 °C

30m
Add Amount50 µL of Concentration10 mg/mL ReagentProteinase KMerck MilliporeSigma (Sigma-Aldrich)Catalog #3115879001

Rotate DurationOvernight at Temperature37 °C

Overnight
Add Amount1 mL of ReagentSodium Chloride, 5 MContributed by users

Shake by hand until evenly mixed
Centrifigation4000 x g, Room temperature, 00:15:00

15m
Transfer supernant to new 1.5mL microcentrifuge tube
Add 2 volumes of Reagent100% EthanolContributed by users

Invert tube to mix until white strings (DNA) begin to aggregate together (approximately 20 times)
Critical
Try to remove the DNA via spooling with a sterile p10 tip and place into a new 1.5mL microcentrifuge tube
If you are unable to remove the DNA with a tip, you can Centrifigation10000 x g, 4°C, 00:05:00 to pellet the DNA

5m
Remove ethanol and air dry pellet on the benchtop for Duration00:05:00

5m
Critical
Resuspend DNA in at least Amount200 µL of Reagent1X TE buffer (10 mM Tris-HCl pH 8.0 1 mM EDTA)Contributed by users buffer Temperature4 °C DurationOvernight

Overnight
If the DNA has the consistency of gelatin after the overnight incubation, you will need to add an addition 200-500uL of Reagent1X TE buffer (10 mM Tris-HCl pH 8.0 1 mM EDTA)Contributed by users to get the DNA to go into solution.

Overnight
Quantify the DNA concentration using your method of choice; we recommend using a Qubit and taking 3 separate readings within the tube to get an average concentration: 1 from the top, 1 from the middle and 1 from the bottom.


in vitro transcription of guide RNA
in vitro transcription of guide RNA
1h 15m
1h 15m
Lyophilized custom DNA oligos for the specific guide RNA (gRNA) were purchased from IDT or Sigma Aldrich and resuspended to Concentration100 micromolar (µM) . A 1:10 dilution was used as a working stock for in vitro transcription.

in vitro transcription of a gRNA was setup as follows using the EnGen sgRNA Synthesis Kit (NEB, E3322S) in a PCR tube:
Amount10 µL 2x EnGen sgRNA Reaction Mix
Amount0.5 µL 100mM DTT
Amount2.5 µL 1:10 custom DNA oligo
Amount2 µL EnGen sgnRNA enzyme mix
Amount5 µL molecular biology grade water

Incubate Temperature37 °C Duration01:00:00

1h
Add Amount30 µL molecular biology grade water to bring the sample to 50uL

Add Amount2 µL DNase to degrade remaining DNA oligos within the sample

Critical
Incubate Temperature37 °C Duration00:15:00

15m
Critical
sgRNA purification
sgRNA purification
55m 15s
55m 15s
Move the in vitro transcribed sgRNA from the PCR tube into a 1.5mL microcentrifuge tube
Add Amount200 µL ReagentTRIzol ReagentThermo Fisher ScientificCatalog #15596026 and Amount50 µL ReagentChloroformContributed by users to the sample.

Toxic
Vortex for Duration00:00:10 to mix thoroughly

10s
Centrifuge Centrifigation20000 x g, Room temperature, 00:05:00

5m
Remove the aqueous layer (should be the top clear layer) carefully, avoiding the middle (should be barely able to see if at all) and bottom (pink) layer, and transfer to a new 1.5mL microcentrifuge tube.
Add Amount50 µL ReagentChloroformContributed by users

Toxic
Vortex for Duration00:00:05 to mix

5s
Centrifuge Centrifigation20000 x g, Room temperature, 00:05:00

5m
Remove the top aqueous layer and place in a new 1.5mL microcentrifuge tube
Add 2 volumes of Reagent100% EthanolContributed by users and Reagent3M Sodium Acetate solutionContributed by users to a final concentration of 0.3M

Invert to mix
Centrifuge Centrifigation20000 x g, 4°C, 00:30:00

30m
Remove the supernant being careful not to disturb the white pellet at the bottom of the tube
Wash the RNA pellet with Amount250 µL 70% ethanol

Centrifuge Centrifigation20000 x g, 4°C, 00:10:00

10m
Repeat 70% ethanol wash and centrifugation step
Remove as much residual ethanol as possible from the RNA pellet
Critical
Air dry on benchtop for Duration00:05:00

5m
Resuspend pellet in Amount10 µL RNase-free water

Quantify the RNA concentration using your method of choice, we recommend and use the Qubit
Cas9 enrichment for Mobile Element Insertions (MEIs)
Cas9 enrichment for Mobile Element Insertions (MEIs)
1h 50m
1h 50m
To perform enrichment for your specific MEI, be sure to use at least 5ug of HMW gDNA at a concentration of around 100ng/uL. For our L1H enrichment experiments, we used 10ug of gDNA.
Add gDNA to a PCR tube and then the following to dephosphorylate the gDNA:
Amount4 µL 1X Cutsmart Buffer
Amount6 µL Quick CIP
AmountX µL molecular grade water to 40uL total

Incubate in the thermocycler using the below protocol:
Temperature37 °C Duration00:30:00
Temperature80 °C Duration00:20:00

50m
Make a 1:5 dilution of the Cas9 protein making the following mix in a PCR tube:
Amount2.5 µL 1X Cutsmart Buffer (make a 1X solution of the 10X buffer to use for this)
Amount0.5 µL Alt-R S.p. Cas9 HiFi V3 nuclease
Mix by flicking the tube
Critical
While the gDNA is being dephosphorylated, make the Cas9 RNP using the mix below in a PCR tube:
Amount850 ng sgRNA
Amount1 µL 1:5 dilution of Cas9 protein
Amount1.5 µL 10X Cutsmart Buffer
AmountX µL molecular biology grade water to 15uL total

Mix thoroughly by inversion
Incubate RNP TemperatureRoom temperature Duration00:20:00

20m
Cool the dephosphorylated gDNA sample on ice

Add Amount15 µL RNP , Amount1 µL Taq DNA polymerase and Amount1.5 µL dATP to the gDNA sample tube

Mix by inversion or flicking
Incubate in a thermocycler using the below protocol:
Temperature37 °C Duration00:30:00 for Cas9 cleavage
Temperature72 °C Duration00:10:00 for a-tailing of cut ends

40m
ONT sequencing on R9 flow cells
ONT sequencing on R9 flow cells
55m
55m
Allow the Cas9-enriched sample to cool on ice then move the sample into a 1.5mL microcentrifuge tube
Add the following from the SQK-LSK109 ONT ligation sequencing kit to the sample tube for sequencing adapter ligation:
Amount25 µL LNB
Amount5 µL AMX
Amount5 µL T4 DNA ligase (NEB, M0202M)
Amount15 µL molecular biology grade water for a total of 100uL

Mix by flicking then spin down briefly
Rotate TemperatureRoom temperature Duration00:30:00

30m
Add 1 volume of Reagent1X TE buffer (10 mM Tris-HCl pH 8.0 1 mM EDTA)Contributed by users and mix by inversion

Critical
Add Amount40 µL Axygen magnetic beads from the SQK-LSK109 kit

Mix by inversion
Rotate TemperatureRoom temperature Duration00:10:00 then TemperatureRoom temperature Duration00:05:00 without rotation

15m
Place tube on magnetic rack and pellet the magnetic beads
Remove the supernant
Add Amount200 µL LFB and resuspend by flicking to wash

Briefly spin down the beads and place on magnetic rack to pellet the beads
Repeat steps 65-67
Spin down the beads once more and place on the magnetic rack, remove as much of the residual LFB as possible
Critical
Add Amount16.8 µL EB and resuspend the beads by flicking the tube

Incubate TemperatureRoom temperature Duration00:10:00

10m
Place tube on magnetic rack and pellet the beads
Remove the elution and put into new 1.5mL microcentrifuge tube, discard the tube with the beads
Quantify the library concentration using your method of choice, we recommend and use the Qubit
Prepare the MinION flow cell for sequencing following ONT's protocol
For loading the library onto the flow cell, add Amount26 µL SQB to the library. We load as much library as we can onto the flow cell.

Immediately before loading onto the flow cell, add Amount0.5 µL SQT and Amount9.5 µL LB to the library.

Load the library onto the MinION following ONT's protocol
Protocol references
Original high molecular weight DNA salting-out protocol: Miller, S. A., Dykes, D. D. & Polesky, H. F. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res. 16, 1215 (1988).

Original nCATS protocol: Gilpatrick, T. et al. Targeted nanopore sequencing with Cas9-guided adapter ligation. Nat. Biotechnol. 38, 433–438 (2020).