Library tissue handling, viral DNA extraction, and NGS sample preparation
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, Miguel Chuapoco
Abstract
This protocol describes the procedure to isolate viral DNA from AAV-transfected tissue and prepare it for next-generation sequencing.
Steps
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
Add 100mg (brain, liver, or spinal cord) and 1mL
Equipment
| Value | Label |
|---|---|
| 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 |
Equipment
| Value | Label |
|---|---|
| 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 |
Homogenize tissue in using the following settings:
- Speed: 5.0 m/s
- Time: 30 seconds
- Pause: 1 minute
- Cycles: 2
Incubate for 0h 5m 0s.
Equipment
| Value | Label |
|---|---|
| BEADBUG 6, SIX POSITION HOMOGENIZER, 115V | NAME |
| Tissue homogenizer (6 position) | TYPE |
| Benchmark Scientific | BRAND |
| D1036 | SKU |
Equipment
| Value | Label |
|---|---|
| BEADBLASTER 24 MICROTUBE HOMOGENIZER, 115V | NAME |
| Tissue Homogenizer (24 position) | TYPE |
| Benchmark Scientific | BRAND |
| D2400 | SKU |
Centrifuge the homogenizer tubes containing the TRIzol solution and homogenized tissue using the following parameters: 12000x g,4°C. Transfer the supernatant to a new tube (microcentrifuge tube or similar).
Equipment
| Value | Label |
|---|---|
| Centrifuge 5425/5425 R - Microcentrifuge | NAME |
| Refrigerated centrifuge | TYPE |
| Eppendorf | BRAND |
| 2231000909 | SKU |
Equipment
| Value | Label |
|---|---|
| DNA LoBind® Tubes | NAME |
| Microcentrifuge tubes | TYPE |
| Eppendorf | BRAND |
| 022431021 | SKU |
Add 600µL to each tube for every 1mL used for lysis, vortex briefly, and incubate for 0h 3m 0s.
Centrifuge 12000x g,4°C to separate the nucleic acid phase from the protein phase. Transfer the top aqueous phase to a new tube (approximately 500µL)
Equipment
| Value | Label |
|---|---|
| Centrifuge 5425/5425 R - Microcentrifuge | NAME |
| Refrigerated centrifuge | TYPE |
| Eppendorf | BRAND |
| 2231000909 | SKU |
Equipment
| Value | Label |
|---|---|
| DNA LoBind® Tubes | NAME |
| Microcentrifuge tubes | TYPE |
| Eppendorf | BRAND |
| 022431021 | SKU |
Add 1 equivalent volume of isopropanol, 1/10 volume of sodium acetate, and co-precipitant (e.g. 500µL, 50µL, 2-3µL) and vortex briefly. Incubate for 0h 10m 0s.
Centrifuge 12000x g,4°C to pellet nucleic acids. Discard supernatant and wash pellet with 1mL. Centrifuge again 7500x g,4°C and discard supernatant.
Air dry pellet and resuspend in 84µL
Remove RNA by digestion with 3µL and digest with 3µL. Supplement reaction with 10µL. Incubate at Room temperature and 37°C.
Purify with
Genome Recovery
Amplify the region around the diversified genome insertion by PCR with 25 cycles of 98°C for 0h 0m 10s, 60°C for 0h 0m 30s and72°C for 0h 30m 0s using
Index addition
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 0h 0m 10s, 59°C for 0h 0m 30s, and 72°C for 0h 0m 10s using
Purify with
Append Illumina flow cell adapters and unique indices by PCR amplification with
by 10 cycles of 98°C for 0h 0m 10s, 59°C for 0h 0m 30s, and 72°C for 0h 0m 10s using
Clean up and validation
Run PCR products on a freshly-prepared 2%
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.
