Nov 19, 2024
Version 1
TEnCATS - Transposable Element nanopore Cas9-Targeted Sequencing V.1
This protocol is a draft, published without a DOI.
- 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 2024. TEnCATS - Transposable Element nanopore Cas9-Targeted Sequencing. Protocol exchange https://protocols.io/view/tencats-transposable-element-nanopore-cas9-targete-drzg573wVersion 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
Created: November 18, 2024
Last Modified: November 22, 2024
Protocol Integer ID: 112392
Keywords: Cas9, nCATS, transposable elements, TE, R10, ONT, SMaHT
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)
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
High Molecular Weight (HMW) genomic DNA (gDNA) extraction from tissue
45m
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 2000rpm
15m
Incubate the tissue sample an additional 00:30:00 at 56 °C without agitation
30m
Continue with Monarch protocol
in vitro transcription of guide RNAs (sgRNAs)
in vitro transcription of guide RNAs (sgRNAs)
1h 15m
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
Purification of sgRNAs
55m
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, Room temperature, 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 thorougly
Centrifuge 20000 x g, Room temperature, 00:05:00
5m
Pipet out the top layer and place into a new 1.5mL microcentrifuge tube
Add 2 volumes of 70% 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 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
Cas9-targeting of transposable elements in HMW gDNA
1h 31m
1h 31m
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 following reaction if needed.
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
Incubate the sample to dephosphorylate the gDNA using the below protocol in a thermocylcer:
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
2 µL Taq DNA polymerase
1.5 µL 10mM dATP
Mix by inversion
Incubate the sample using the protocol below in a thermocycler:
37 °C 00:30:00
72 °C 00:10:00
40m
ONT library preparation for R10 MinION flow cells
ONT library preparation for R10 MinION flow cells
56m
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 flicking
Incubate Room temperature 00:30:00 with rotation
30m
Add 1 volume of 1X TE buffer and invert to mix
Add 40 µL Axygen magnetic beads to the sample and invert to mix
Incubate Room temperature 00:10:00 with rotation
10m
Incubate Room temperature 00:05:00 without rotation
5m
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 beads on magnetic rack and removing the supernant
Remove as much residual LFB as you can
Add 13 µL EB to the sample and resuspend the beads in the buffer
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
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 SQB
25.5 µL LB
Prime the MinION following ONT's instructions
Mix the library to resuspend the LB and load onto the flow cell following ONT's instructions