May 23, 2023
Version 2
Library tissue handling, viral DNA extraction, and NGS sample preparation V.2
Forked from a private protocol
- Miguel Chuapoco1,
- Sripriya Ravindra Kumar1,
- Timothy F. Shay1,
- Xinhong Chen1,
- David Brown1,
- Tatyana Dobreva1,
- Qin Huang1,
- Xiaozhe Ding1,
- Yicheng Luo1,
- Pétur H. Einarsson1,
- Alon Greenbaum1,
- Min J. Jang1,
- Benjamin E. Deverman1,
- Viviana Gradinaru1
- 1California Institute of Technology
External link: https://rdcu.be/c9RyP
Protocol Citation: Miguel Chuapoco, Sripriya Ravindra Kumar, Timothy F. Shay, Xinhong Chen, David Brown, Tatyana Dobreva, Qin Huang, Xiaozhe Ding, Yicheng Luo, Pétur H. Einarsson, Alon Greenbaum, Min J. Jang, Benjamin E. Deverman, Viviana Gradinaru 2023. Library tissue handling, viral DNA extraction, and NGS sample preparation. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l695zklqe/v2Version created by Miguel Chuapoco
Manuscript citation:
Ravindra Kumar, S., Miles, T.F., Chen, X.et al.Multiplexed Cre-dependent selection yields systemic AAVs for targeting distinct brain cell types.Nat Methods17, 541–550 (2020). https://doi.org/10.1038/s41592-020-0799-7
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: May 23, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 82290
Keywords: ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson’s
Grant ID: ASAP-020495
Abstract
This protocol describes the procedure to isolate viral DNA from AAV-transfected tissue and prepare it for next-generation sequencing.
Protocol materials
UltraPure™ Low Melting Point AgaroseThermo Fisher ScientificCatalog #16520050
Step 19
RNase Cocktail™ Enzyme MixThermo FisherCatalog #AM2286
Step 13
NEBNext Multiplex Oligos for Illumina (Dual Index Primers Set 1) - 96 rxnsNew England BiolabsCatalog #E7600S
Step 18
CutSmart Buffer - 5.0 mlNew England BiolabsCatalog #B7204S
Step 13
Zymo DNA Clean & Concentrator - 5Zymo ResearchCatalog #D4014
In 2 steps
Q5 High-Fidelity DNA Polymerase - 500 unitsNew England BiolabsCatalog #M0491L
In 3 steps
Sodium Acetate 3M, pH 5.2Thermo ScientificCatalog #R1181
Step 10
GlycoBlue™ CoprecipitantThermo ScientificCatalog #AM9516
Step 10
UltraPure Distilled Water Invitrogen - Thermo FisherCatalog #10977-015
Step 12
TRIzol ReagentThermo Fisher ScientificCatalog #15596026
Step 5
SmaI - 2,000 unitsNew England BiolabsCatalog #R0141S
Step 13
Marmoset tissue extraction (library selections)
Marmoset tissue extraction (library selections)
Raise and house marmosets in compliance with your local Institutional Animal Care and Use Committee (IACUC) and ensure that all protocols and procedures have been approved by the appropriate ethical and regulatory committees.
For adult marmosets, ensure that there is no detectable neutralizing antibodies for AAV9. This can be conducted by The Penn Vector Core at the University of Pennsylvania (https://gtp.med.upenn.edu/intranethome/core-facilities-internal/immunology-core)
Inject marmosets with desired dose of library (e.g. 2 x 1012 vector genomes of library) intravenously (e.g. via the femoral vein).
At four weeks post-injection, euthanize marmosets and perfuse with 1X phosphate buffered saline.
Flash freeze tissue (e.g. using 2-methylbutane chilled with dry ice). Separate the brain into coronal blocks and flash freeze the blocks. Store tissue at -80 ºC until ready for processing.
DNA Extraction
DNA Extraction
Add 100 mg tissue sample (brain, liver, or spinal cord) and 1 mL TRIzol reagent TRIzol ReagentThermo Fisher ScientificCatalog #15596026 to bead homogenizer tubes. Use prefilled tubes with 1.5 mm Zirconium beads or 2.8 mm stainless steel beads.
Equipment
Prefilled 2.0ml tubes, Zirconium Beads, 1.5mm Triple-Pure - High Impact, 50pk
NAME
Homogenizer tubes (1.5 mm Zirconium beads)
TYPE
Benchmark Scientific
BRAND
D1032-15
SKU
LINK
Equipment
Prefilled 2.0ml tubes, Stainless Steel, 2.8mm Acid Washed, 50pk
NAME
Homogenizer tubes (2.8 stainless steel)
TYPE
Benchmark Scientific
BRAND
D1033-28
SKU
LINK
Homogenize tissue in using the following settings:
- Speed: 5.0 m/s
- Time: 30 seconds
- Pause: 1 minute
- Cycles: 2
Incubate for 00:05:00 .
Equipment
BEADBUG 6, SIX POSITION HOMOGENIZER, 115V
NAME
Tissue homogenizer (6 position)
TYPE
Benchmark Scientific
BRAND
D1036
SKU
LINK
Equipment
BEADBLASTER 24 MICROTUBE HOMOGENIZER, 115V
NAME
Tissue Homogenizer (24 position)
TYPE
Benchmark Scientific
BRAND
D2400
SKU
LINK
Note
Samples can be stored at -20 ºC for up to year in TRIzol.
5m
Centrifuge the homogenizer tubes containing the TRIzol solution and homogenized tissue using the following parameters: 12000 x g, 4°C, 00:05:00 . Transfer the supernatant to a new tube (microcentrifuge tube or similar).
Equipment
Centrifuge 5425/5425 R - Microcentrifuge
NAME
Refrigerated centrifuge
TYPE
Eppendorf
BRAND
2231000909
SKU
LINK
Equipment
DNA LoBind® Tubes
NAME
Microcentrifuge tubes
TYPE
Eppendorf
BRAND
022431021
SKU
LINK
5m
Add 600 µL chloroform to each tube for every 1 mL TRIzol used for lysis, vortex briefly, and incubate for 00:03:00 .
3m
Centrifuge 12000 x g, 4°C, 00:15:00 to separate the nucleic acid phase from the protein phase. Transfer the top aqueous phase to a new tube (approximately 500 µL aqueous phase )
Equipment
Centrifuge 5425/5425 R - Microcentrifuge
NAME
Refrigerated centrifuge
TYPE
Eppendorf
BRAND
2231000909
SKU
LINK
Equipment
DNA LoBind® Tubes
NAME
Microcentrifuge tubes
TYPE
Eppendorf
BRAND
022431021
SKU
LINK
15m
Add 1 equivalent volume of isopropanol, 1/10 volume of sodium acetate, and co-precipitant (e.g. 500 µL isopropanol , 50 µL sodium acetate , 2-3 µL co-precipitant ) and vortex briefly. Incubate for 00:10:00 .
Sodium Acetate 3M, pH 5.2Thermo ScientificCatalog #R1181
GlycoBlue™ CoprecipitantThermo ScientificCatalog #AM9516
10m
Centrifuge 12000 x g, 4°C, 00:10:00 to pellet nucleic acids. Discard supernatant and wash pellet with 1 mL 75% ethanol . Centrifuge again 7500 x g, 4°C, 00:05:00 and discard supernatant.
15m
Air dry pellet and resuspend in 84 µL PCR clean water
UltraPure Distilled Water Invitrogen - Thermo FisherCatalog #10977-015
Remove RNA by digestion with 3 µL RNase cocktail and digest with 3 µL SmaI . Supplement reaction with 10 µL CutSmart . Incubate at Room temperature for 2-3 hours and 37 °C overnight .
RNase Cocktail™ Enzyme MixThermo FisherCatalog #AM2286
SmaI - 2,000 unitsNew England BiolabsCatalog #R0141S
CutSmart Buffer - 5.0 mlNew England BiolabsCatalog #B7204S
Purify with Zymo DNA Clean & Concentrator - 5Zymo ResearchCatalog #D4014
Genome Recovery
Genome Recovery
30m 40s
30m 40s
Amplify the region around the diversified genome insertion by PCR with 25 cycles of 98 °C for 00:00:10 , 60 °C for 00:00:30 and72 °C for 00:30:00 using Q5 High-Fidelity DNA Polymerase - 500 unitsNew England BiolabsCatalog #M0491L and 50% of the total extracted viral DNA as a template using primers XF (ACTCATCGACCAATACTTGTACTATCTCTCTAGAAC) and 588-R2lib-R (GTATTCCTTGGTTTTGAACCCAACCG).
30m 40s
Index addition
Index addition
1m 40s
1m 40s
Dilute PCR product 1:100 and use as template for an additional round of PCR amplification around the variable region with primers containing Read1 and Read2 sequences by 10 cycles of 98 °C for 00:00:10 , 59 °C for 00:00:30 , and 72 °C for 00:00:10 using Q5 High-Fidelity DNA Polymerase - 500 unitsNew England BiolabsCatalog #M0491L and primers 588i-lib-PCR1-6bpUID-F (CACTCATCGACCAATACTTGTACTATCTCTCT) and 588i-lib-PCR1-R (GTATTCCTTGGTTTTGAACCCAACCG).
50s
Purify with Zymo DNA Clean & Concentrator - 5Zymo ResearchCatalog #D4014
Append Illumina flow cell adapters and unique indices by PCR amplification with NEBNext Multiplex Oligos for Illumina (Dual Index Primers Set 1) - 96 rxnsNew England BiolabsCatalog #E7600S
by 10 cycles of 98 °C for 00:00:10 , 59 °C for 00:00:30 , and 72 °C for 00:00:10 using Q5 High-Fidelity DNA Polymerase - 500 unitsNew England BiolabsCatalog #M0491L
50s
Clean up and validation
Clean up and validation
Run PCR products on a freshly-prepared 2% UltraPure™ Low Melting Point AgaroseThermo Fisher ScientificCatalog #16520050 gel.
Verify the expected size of the band and extract from the gel.
If desired, verify the nucleotide diversity at the randomized insertion site by Sanger sequencing.
Note
If additional material is needed for Sanger sequencing, perform an additional PCR amplification using 15-20 cycles of 98 °C for 00:00:10 , 60 °C for 00:00:30 and72 °C for 00:00:10 with primers NGS-QC-F (AATGATACGGCGACCACCGAG) and NGS-QC-R (CAAGCAGAAGACGGCATACGA).