Aug 29, 2025
Version 2
TEnCATS - Transposable Element nanopore Cas9-Targeted Sequencing V.2
- Torrin McDonald1,
- Camille Mumm1,
- Jessica Switzenberg1,
- Alan Boyle2
- 1University of Michigan;
- 2University of Michigan - Ann Arbor
Protocol Citation: Torrin McDonald, Camille Mumm, Jessica Switzenberg, Alan Boyle 2025. TEnCATS - Transposable Element nanopore Cas9-Targeted Sequencing. protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg3q66ev25/v2Version created by Jessica Switzenberg
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 currently for tissue and cell line capture experiments.
Created: August 28, 2025
Last Modified: August 29, 2025
Protocol Integer ID: 225710
Keywords: Cas9, nCATS, transposable elements, TE, R10, ONT, SMaHT, transposable elements within genomic dna, using oxford nanopore technology, oxford nanopore technology, using cas9, r10 flow cell, genomic dna, cas9, dna, boyle lab, transposable element
Abstract
This is the Boyle lab's protocol for using Cas9 to target transposable elements within genomic DNA using Oxford Nanopore Technologies (ONT) R10 flow cells.
Materials
Monarch HMW DNA Extraction Kit for Tissue (T3060S, NEB)
Proteinase K (3115879001, Roche)
Thermomixer
EnGen sgRNA Synthesis kit (E3322S, NEB)
Molecular biology grade water
Trizol
Chloroform
Ethanol
3M sodium acetate
Refrigerated centrifuge
RNase-free water
Qubit with BR RNA assay kit, dsDNA BR assay kit, dsDNA HS assay kit
Alt-R S.p. Cas9 HiFi Nuclease V3 (IDT)
QuickCIP (M0525S, NEB)
Taq DNA polymerase (M0273L, NEB)
10mM dATP
T4 DNA ligase (M0202M, NEB)
Thermolabile Proteinase K (P8111S, NEB)
1X TE buffer
SQK-LSK114 kit (ONT)
MinION R10 flow cells (ONT)
MinION sequencer (ONT)
Tube rotator
1.5mL microcentrifuge tubes
PCR tubes
High Molecular Weight (HMW) genomic DNA (gDNA) extraction from tissue
45m
Aliquot the desired amount of tissue for gDNA extraction
Follow the manufacturer's guidelines from the Monarch HMW DNA Extraction Kit for Tissues (T3060S, NEB) with following changes to the lysis step:
Add 40 µL 10 mg/mL Proteinase K to 580 µL Tissue Lysis Buffer
Incubate tissue sample in Tissue Lysis Buffer 56 °C 00:15:00 in Thermomixer at 1000rpm
15m
Incubate the tissue sample an additional 00:30:00 at 56 °C without agitation. If clumps are still visible, you can incubate longer including overnight
30m
Continue with Monarch protocol
in vitro transcription of guide RNAs (sgRNAs)
1h 15m
Lyophilized custom DNA oligos of the desired guide RNAs were resuspended in molecular biology grade water to a final concentration of 100uM. A 1:10 dilution of the oligos was used as a working stock for in vitro transcription
For each guide, set up the below mix in a PCR tube using the EnGen sgRNA Synthesis Kit (E3322S, NEB):
10 µL 2x sgRNA Reaction Mix
0.5 µL DTT
2.5 µL custom DNA oligo
2 µL EnGen Enzyme Mix
5 µL molecular biology grade water
Incubate the reaction 37 °C 01:00:00 in a thermocycler
1h
Add 30 µL molecular biology grade water then 2 µL DNase I to the reaction to remove the DNA oligos
Incubate the reaction 37 °C 00:15:00
15m
Purification of sgRNAs
55m
Move the sgRNAs produced from the in vitro transcription reactions into a new 1.5mL microcentrifuge tube
Add 200 µL Trizol and 50 µL Chloroform to the samples and vortex to mix thoroughly
Centrifuge 20000 x g, 4°C, 00:05:00
5m
Pipet out the aqueous layer (should be clear) carefully to avoid the pink layer and place into a new 1.5mL microcentrifuge tube
Add 50 µL Chloroform and vortex to mix thoroughly
Centrifuge 20000 x g, 4°C, 00:05:00
5m
Pipet out the top layer carefully to avoid the second layer and place into a new 1.5mL microcentrifuge tube
Add 2 volumes of cold 100% ethanol and 3M sodium acetate solution to a final concentration of 0.3M and invert to mix 10 times
Centrifuge 20000 x g, 4°C, 00:30:00
30m
Remove supernant being careful not to disturb the white RNA pellet at the bottom
Wash the sgRNA by adding cold 250 µL 70% ethanol and centrifuging 20000 x g, 4°C, 00:10:00 then removing the supernant. Repeat this wash for a total of 2 washes
10m
Remove as much of the residual ethanol as possible and air dry on bench for 00:05:00
5m
Resuspend the sgRNA in 11 µL RNase-free water
Quantify the RNA concentration using your specific method, we used the Qubit
Cas9-targeting of transposable elements in HMW gDNA
2h 1m
Aliquot out 10ug of extracted gDNA using a wide-bore tip into a PCR tube, try to get this to below 30uL. If not, you can scale up the Cutsmart Buffer and water in the following reaction if needed. Usually when scaling, I try to stay at a maximum volume of 100uL total.
Add the following to the gDNA sample:
4 µL 10X Cutsmart Buffer
6 µL QuickCIP
X µL molecular biology grade water to a total of 40uL.
Mix by inversion then spin down briefly
Incubate the sample to dephosphorylate the gDNA using the below protocol in a thermocycler:
37 °C 00:30:00
80 °C 00:20:00
50m
Make a 1:5 dilution of the Cas9 nuclease as follows:
2.5 µL 1X Cutsmart Buffer
0.5 µL Alt-R S.p Cas9 HiFi nuclease
While the gDNA is dephosphorylating, make the Cas9:sgRNA (RNP) by mixing the following in a PCR tube:
850 ng sgRNA
1 µL 1:5 dilution of Cas9 nuclease
1.5 µL 10X Cutsmart Buffer
X µL RNase-free water to a total of 15uL
Mix by flicking and incubate Room temperature 00:20:00
Cool the dephosphorylated gDNA on ice for 00:01:00
1m
Add the following to the gDNA sample for Cas9 cleavage and a-tailing of open ends:
15 µL RNP
1.5 µL Taq DNA polymerase
1 µL 10mM dATP
Mix by inversion and spin down briefly
Incubate the sample using the protocol below in a thermocycler:
37 °C 00:30:00
72 °C 00:10:00
40m
Cool the Cas9-cleaved gDNA mix for 00:01:00 on ice
Add 5 µL TL Proteinase K (P8111S, NEB) to the sample
Mix by inversion and spin down briefly
Incubate the sample using the protocol below in a thermocycler:
37 °C 00:20:00
72 °C 00:10:00
30m
ONT library preparation for R10 MinION flow cells
56m
Cool the Cas9-treated sample on ice for 00:01:00
1m
Transfer the sample to a new 1.5mL microcentrifuge tube using a wide-bore tip
Add the following to the sample from the ONT SQK-LSK114 kit:
25 µL LNB
5 µL LA
then add 5 µL T4 DNA ligase (M0202M, NEB) and X µL molecular biology grade to 100uL total and mix by inversion, then spin down briefly
*Note: If you scaled up your dephosphorylation reaction, you will need to scale the amount of LNB here to be 1/4 of the total reaction volume i.e. 125uL of sample (dephos gDNA reaction+RNP+Taq+dATP+LA+T4 ligase) would be 31.25uL of LNB*
Incubate Room temperature 00:30:00 with rotation with a tube rotator
30m
Add 1 volume of 1X TE buffer and invert to mix
Add 80 µL Axygen magnetic beads to the sample and invert to mix
*Note: If you scaled up your reaction, you will need to add the beads to be a final of 0.4x i.e. 250uL (Reaction volume in Step 33 + 1x TE) is 100uL of beads*
Incubate Room temperature 00:10:00 with rotation
10m
Place tube on a magnetic rack and pellet the beads
Remove supernant
Wash beads twice by adding 200 µL LFB , resuspending the beads by inversion, pellet the beads on the magnetic rack and remove the supernant
Remove as much residual LFB as you can by spinning the tube down briefly and pelleting the bead on the magnetic rack, and pipeting out any leftover LFB
Add 13 µL EB to the sample and resuspend the beads in the buffer. I have also brought this up to 16uL of EB due to scaling up and used 12uL of this library for sequencing
Incubate 37 °C 00:10:00
10m
Place tube on magnetic rack and pellet the beads
Pipet out the eluate and place in new 1.5mL microcentrifuge tube
Quantify the library using your method of choice, we use the Qubit
Loading onto the R10 MinION flow cell
For sequencing, we load as much library as we can onto the flow cell thus the entire 12uL of library is used. Add the following to the library:
37.5 µL SB
25.5 µL LIB
Prime the MinION following ONT's instructions
Mix the library by quick inversion or flicking to resuspend the LIB and load onto the flow cell following ONT's instructions