TITLE: AAV viral DNA and whole RNA recovery for AAV pool experiments in rhesus macaque
AUTHORS: Miguel Chuapoco

[DESCRIPTION]
This protocol outlines procedures to extract viral DNA and whole RNA from rhesus macaque tissue that had been treated with AAV in vivo

[STEPS]
SECTION: DNA/RNA extraction
1. Add 100 mg (brain or liver) 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/RNA extraction
2. 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/RNA extraction
3. 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/RNA extraction
4. Add 200 µL to each tube for every 1 mL used for lysis, vortex briefly, and incubate for 3 min. 
SECTION: DNA/RNA extraction
5. 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/RNA extraction
6. 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/RNA extraction
7. Air dry pellet and resuspend in 84 µL 
  
SECTION: DNA/RNA extraction
8. To isolate DNA, treat half of the sample with RNase. Remove RNA by digestion with 1.5 µL and digest with 1.5 µL. Supplement reaction with 5 µL. Incubate at Room temperature and 37 °C.

Purify with   

  
  
  
SECTION: DNA/RNA extraction
9. Incubate 65 °C 5 min and place on ice.

SECTION: DNA/RNA extraction
8.1. To obtain cDNA, take 1 µg from sample (measured by high sensitivity RNA Qubit) and treat with  


Combine  1 µg with 1 µL, 1 µL, and   to 10 uL. 

Incubate reaction Room temperature 15 min 

SECTION: DNA/RNA extraction
8.2. Inactivate DNase I by adding 1 µL. Heat 65 °C15 min 

SECTION: DNA/RNA extraction
8.3. To convert DNase I treated RNA to cDNA, take 1-5 µL and combine with 1 µL , 1 µL and fill to 10 µL using UltraPure water.


  

SECTION: DNA/RNA extraction
10. Prepare cDNA synthesis mix according to manufacturer's specifications. Add 10 µL to each RNA primer mixture and mix by gently flicking the tubes. 

SECTION: DNA/RNA extraction
11. Incubate as follows: 
50 °C 50 min 
25 °C 10 min 
50 °C 50 min 
85 °C 5 min 
SECTION: DNA/RNA extraction
12. Store samples at -80 °C until ready to use. 
SECTION: PCR amplification
13. Use Zymo DNA purification of PCR product according to manufacturer's suggested protocol.  
SECTION: PCR amplification
14. 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, 60 °C for 30 s, and 72 °C for 10 s using   and the following primers: 
Forward: 5'- TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGcgatgttccagattacgcttgag -3'
Reverse: 5'- GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGattttgtaatccagaggttgattatcg - 3'

SECTION: PCR amplification
15. Use Zymo DNA purification of PCR product according to manufacturer's suggested protocol.  
SECTION: PCR amplification
16. 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: PCR amplification
17. Run PCR products on a freshly-prepared 2%   gel and gel purify amplified 200 bp PCR product.