TITLE: Library tissue handling, viral DNA extraction, and NGS sample preparation
AUTHORS: 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

[DESCRIPTION]
This protocol describes the procedure to isolate viral DNA from AAV-transfected tissue and prepare it for next-generation sequencing.

[STEPS]
SECTION: DNA Extraction
5. Add 100 mg (brain, liver, or spinal cord) and 1 mL  to bead homogenizer tubes.  Use prefilled tubes with 1.5 mm Zirconium beads or 2.8 mm stainless steel beads. 
 

 

SECTION: DNA Extraction
13. Remove RNA by digestion with 3 µL and digest with 3 µL. Supplement reaction with 10 µL. Incubate at Room temperature and 37 °C.

  
  
  

SECTION: DNA Extraction
6. Homogenize tissue in using the following settings: 
Speed: 5.0 m/s 
Time: 30 seconds 
Pause: 1 minute
Cycles: 2

Incubate for 5 min.
 

 

 




SECTION: DNA Extraction
14. Purify with   

SECTION: Genome Recovery
15. Amplify the region around the diversified genome insertion by PCR with 25 cycles of 98 °C for 10 s, 60 °C for 30 s and72 °C for 30 min using   and 50% of the total extracted viral DNA as a template using primers XF (ACTCATCGACCAATACTTGTACTATCTCTCTAGAAC) and 588-R2lib-R (GTATTCCTTGGTTTTGAACCCAACCG).

SECTION: Index addition
16. 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 10 s, 59 °C for 30 s, and 72 °C for 10 s using   and primers 588i-lib-PCR1-6bpUID-F (CACTCATCGACCAATACTTGTACTATCTCTCT) and 588i-lib-PCR1-R (GTATTCCTTGGTTTTGAACCCAACCG).
SECTION: Index addition
17. Purify with   

SECTION: Index addition
18. Append Illumina flow cell adapters and unique indices by PCR amplification with   
by 10 cycles of 98 °C for 10 s, 59 °C for 30 s, and 72 °C for 10 s using  
SECTION: Clean up and validation
19. Run PCR products on a freshly-prepared 2%   gel.

SECTION: Clean up and validation
20. Verify the expected size of the band and extract from the gel.
SECTION: Clean up and validation
21. If desired, verify the nucleotide diversity at the randomized insertion site by Sanger sequencing. 
 

SECTION: DNA Extraction
7. Centrifuge the homogenizer tubes containing the TRIzol solution and homogenized tissue using the following parameters:  12000 x g, 5 min, 4 °C. Transfer the supernatant to a new tube (microcentrifuge tube or similar). 
 

 


SECTION: DNA Extraction
8. Add 600 µL to each tube for every 1 mL used for lysis, vortex briefly, and incubate for 3 min. 

SECTION: DNA Extraction
9. Centrifuge  12000 x g, 15 min, 4 °C to separate the nucleic acid phase from the protein phase. Transfer the top aqueous phase to a new tube (approximately 500 µL)
 

 


SECTION: DNA Extraction
10. Add 1 equivalent volume of isopropanol, 1/10 volume of sodium acetate, and co-precipitant (e.g. 500 µL, 50 µL, 2-3 µL) and vortex briefly. Incubate for 10 min. 

  
  

SECTION: DNA Extraction
11. Centrifuge  12000 x g, 10 min, 4 °C to pellet nucleic acids. Discard supernatant and wash pellet with 1 mL. Centrifuge again 7500 x g, 5 min, 4 °C and discard supernatant.

SECTION: DNA Extraction
12. Air dry pellet and resuspend in 84 µL 
  

SECTION: Marmoset tissue extraction (library selections)
1. 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)
SECTION: Marmoset tissue extraction (library selections)
2. Inject marmosets with desired dose of library (e.g. 2 x 1012 vector genomes of library) intravenously (e.g. via the femoral vein). 
SECTION: Marmoset tissue extraction (library selections)
3. At four weeks post-injection, euthanize marmosets and perfuse with 1X phosphate buffered saline.
SECTION: Marmoset tissue extraction (library selections)
4. 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.