TITLE: AAV-Zombie on cultured cells
AUTHORS: Gerard Michael Coughlin

[DESCRIPTION]
Detection of AAV genomes in situ can facilitate understanding of AAV transduction and processing. This protocol enables detection of AAV genomes and concatemers in cultured cells and is based on the Zombie method published in Askary et al., Nat Biotechnol 38: 66. AAVs carrying barcoded genomes are delivered to cells. Following fixation, a phage RNA polymerase is used to transcribe the barcode in situ, yielding barcoded transcripts that can be detected with RNA FISH. This protocol is compatible with either ssAAV or scAAV genomes. In the former case, detected genomes will be primarily nuclear and detectable once second strand synthesis has occurred. In the latter case, the genome can be detected regardless of where in the transduction pathway the genome is. This protocol can be combined with RNA FISH detection of reporter transcripts or endogenous transcripts, enabling measurement of expression and cell type classification.

[BEFORE_START]
This protocol enables spatial detection of AAV genomes and concatemers in cultured cells. For detection of AAV genomes, a Zombie barcode and phage RNA polymerase promoter are required in a single AAV genome. For detection of AAV concatemers (SpECTr), 2 AAV genomes are required: one genome carries a Zombie barcode and the other carries a phage RNA polymerase promoter. Barcode sequences can be found in Supplementary Table 1 of Askary et al., Nat Biotechnol 38: 66.

See https://www.biorxiv.org/content/10.1101/2023.12.23.573214v1 for more information on these methods. 

[STEPS]
SECTION: Reagent set up
1. General note on reagents and consumables

 

SECTION: Sample preparation
16. At time of collection, aspirate media and wash cells one time with 1 mL  DPBS. Aspirate DPBS and add 1 mL ice-cold MAA fixation buffer. Transfer to -20 °C for 15 min . 

 

SECTION: Sample preparation
13. Transduce cells with AAVs carrying Zombie barcode and phage RNA polymerase promoter (barcode sequences can be found in Supplementary Table 1 of Askary et al., Nat Biotechnol 38: 66).

 

SECTION: In situ transcription and probe hybridization
18. General note on scaling reactions

 

SECTION: Reagent set up
2. Prepare 3:1 methanol:acetic acid (MAA) fixation buffer by combining:

Methanol: 30 mL 
Glacial acetic acid: 10 mL 

in a 50 mL conical tube. Store solution at -20 °C  for up to 3 months.

 

SECTION: Reagent set up
3. Prepare 70% ethanol solution, by combining

100% Ethanol: 35 mL 
UltraPure water: 15 mL 

in a 50 mL conical tube. Store solution at -20 °C for up to 3 months.

 

SECTION: Reagent set up
5. Prepare probe hybridization buffer, by combining:

20x saline-sodium citrate (SSC) buffer: 5 mL 
Ethylene carbonate: 5 mL 
50% dextran sulfate: 10 mL 
UltraPure water: 30 mL 

in a 50 mL conical tube. Store solution at Room temperature for up to 3 weeks, or -20 °C for up to a year. 

 

SECTION: Reagent set up
6. Prepare stringent wash buffer, by combining:

20x saline-sodium citrate (SSC) buffer: 5 mL 
Ethylene carbonate: 15 mL 
UltraPure water: 30 mL 

in a 50 mL conical tube. Store solution at Room temperature for up to 3 weeks, or -20 °C for up to a year. 

 

SECTION: Reagent set up
4. Prepare 4% paraformaldehyde, 1x PBS solution (4% PFA, 1x PBS), by combining:

32% paraformaldehyde: 6.25 mL 
10x Phosphate-buffered saline (PBS): 5 mL 
UltraPure water: 38.75 mL 

in a 50 mL conical tube. Store solution at 4 °C for up to 1 month. 
SECTION: Reagent set up
7. Prepare wash buffer (5x SSC, with 0.1% Tween-20), by combining:

20x saline-sodium citrate (SSC) buffer: 100 mL 
10% Tween-20: 5 mL 
UltraPure water: 395 mL 

Store solution at Room temperature for up to a year.

SECTION: Reagent set up
9. Prepare HCR amplification buffer, by combining:

20x saline-sodium citrate (SSC) buffer: 5 mL 
Ethylene carbonate: 5 mL 
UltraPure water: 35 mL 

in a 50 mL conical tube. Store solution at Room temperature for up to 3 months.

 

SECTION: Reagent set up
10. Prepare 1x PBS, by adding:

10x PBS: 55.5 mL 

To a 500 mL bottle of UltraPure water. 

SECTION: Sample preparation
12. General note on cell culture.

 

SECTION: Sample preparation
15. (Optional) Dense adherent cultures can complicate cell segmentation and downstream image analysis. It may be beneficial to passage cells to a lower confluence (e.g. 10%) before fixation. 

 

SECTION: Sample preparation
14. (Optional) Depending on cell type, experimental question, AAV genome format, etc., it may be necessary to change the media some time post-transduction (e.g. >3 hr). Between aspiration of the old media and addition of the new media, we recommend doing at least 3 washes in a suitable buffer or media.

 

SECTION: Sample preparation
17. Aspirate MAA into an appropriate container for disposal. Wash cells in 1 time with 1 mL  ice-cold 70% ethanol. Remove ethanol wash and dispose of with MAA. Add 1 mL of ice-cold 70% ethanol and transfer samples to -20 °C until processing. 

 


SECTION: In situ transcription and probe hybridization
19. Prepare the reaction chamber. Remove the wafer from a P1000 pipette tip box, add 50 mL  of MilliPore (or comparable) water to the bottom, and then replace the wafer. Cut an appropriately sized piece of parafilm and lay it over the top of the wafer with the clean side up (i.e. the side that was facing the paper).

Place the box into a 37 °C incubator, for at least 60 min .

 

SECTION: In situ transcription and probe hybridization
20. Thaw the NTP(s) and buffer at Room temperature , then place on ice.  

 

SECTION: In situ transcription and probe hybridization
21. Set up in situ transcription reaction solution. For each coverslip, prepare 20 µL of reaction solution, consisting of:

NTPs: 8 µL (or 2 µL of each NTP, if storing separately)
UltraPure water: 8 µL 
RNA polymerase: 2 µL 
Buffer: 2 µL 

Store reaction solution on ice until use.

 

SECTION: In situ transcription and probe hybridization
22. Retrieve samples from -20 °C storage. Aspirate the 70% ethanol off, briefly wash samples with 1 mL  of 1x PBS. Aspirate wash, add 1 mL of fresh 1x PBS and keep at Room temperature until use. 
SECTION: In situ transcription and probe hybridization
23. Set up tools and disposables. You will need a Kimwipe, a pair of clean forceps (suitable for lifting and manipulating coverslips), a 23g needle with the tip bent (to facilitate lifting of coverslip from the well), and a 15 mL tube with 15 mL of UltraPure water. 
SECTION: In situ transcription and probe hybridization
24. Start in situ transcription reactions.

CRITICAL For these steps, pay careful attention to which side of the coverslip has the cells. 

Remove reaction chamber from 37 °C incubator. Pipette 20 µL of in situ transcription solution onto the surface of the parafilm (if doing multiple coverslips, prepare up to 4 spots at a time; ensure spots are adequately spaced)
Gently lift coverslip from well, using hooked needle and forceps.
Briefly dip coverslip in UltraPure water to remove residual PBS, then gently touch the edge of the coverslip to a Kimwipe to wick away excess liquid
Carefully lower coverslip with cell-side down onto a droplet of the transcription solution, being careful not to trap bubbles underneath
Repeat for each coverslip
SECTION: In situ transcription and probe hybridization
25. Transfer reaction chamber to 37 °C incubator, and incubate transcription reaction for 2-3 hours.

 

SECTION: In situ transcription and probe hybridization
26. During in situ transcription reaction, pre-warm the hybridization buffer to 42 °C . You will need 1 mL of hybridization buffer per coverslip.

 

SECTION: In situ transcription and probe hybridization
27. Following in situ transcription reaction, use forceps to lift coverslips off parafilm and place into a clean 24-well plate, with the cell-side up. Add 500 µL of 4% PFA, 1x PBS solution. Fix cells for 15 min at Room temperature . Following fixation, aspirate PFA solution, then wash twice with 1 mL of 1x PBS, followed by 2 washes in 5x SSC (5 min per wash).

 

SECTION: In situ transcription and probe hybridization
28. After PFA fixation, remove 5x SSC and add 500 µL of pre-warmed hybridization buffer. Incubate at 37 °C for 30 min . 

SECTION: In situ transcription and probe hybridization
29. During incubation in Step 26, prepare probe solution by diluting probes to 2 nM in the remaining hybridization buffer. Return to 42 °C until use. 

 

SECTION: In situ transcription and probe hybridization
30. Following equilibration in hybridization buffer, aspirate old hybridization buffer from samples and add 500 µL of probe solution (Step 27). Wrap seal edge of the plate with parafilm to prevent evaporation. Incubate at 37 °C   .

SECTION: Probe washes and Hybridization chain reaction (HCR) amplification
31. Note on reaction volumes

 

SECTION: Probe washes and Hybridization chain reaction (HCR) amplification
32. Pre-warm stringent wash buffer to 37 °C , vortexing a couple times to ensure components are thoroughly mixed. You will need 1.5 mL of stringent wash buffer per coverslip. 

SECTION: Reagent set up
8. Prepare 5x SSC, by combining:

20x saline-sodium citrate (SSC) buffer: 12.5 mL 
UltraPure water: 37.5 mL 

Store solution at Room temperature for up to a year

SECTION: Probe washes and Hybridization chain reaction (HCR) amplification
33. Once stringent wash buffer is warmed to 37 °C and mixed, wash samples 3 times in stringent wash buffer. Perform 1 brief rinse with 500 µL of buffer, followed by two longer washes with 1 mL  at 37 °C for 30 min each. 

 

SECTION: Probe washes and Hybridization chain reaction (HCR) amplification
34. Following stringent washes, wash 3 times in wash buffer (5x SSC, with 0.1% Tween-20). Perform 1 brief rinse with 1 mL of buffer, followed by two longer washes with 1 mL  at 37 Room temperature for 30 min each. 
SECTION: Probe washes and Hybridization chain reaction (HCR) amplification
35. Following washes, aspirate wash buffer from samples and add 1 mL of HCR amplification buffer. Equilibrate samples in HCR amplification buffer for 30 min at Room temperature .

SECTION: Probe washes and Hybridization chain reaction (HCR) amplification
36. During HCR amplification buffer equilibration step, prepare HCR hairpins.

Aliquot each hairpin separately into PCR tubes
Using a thermocycler, heat hairpins to 95 °C for 2 min 
Remove hairpins from thermocycler and allow to cool back down to room temperature in the dark (at least 10 min 

 

SECTION: Probe washes and Hybridization chain reaction (HCR) amplification
37. Following equilibration in HCR amplification buffer, prepare HCR amplification solution. For each coverslip, prepare 300 µL of amplification solution. Dilute snap-cooled hairpins to 30 nM in the HCR amplification buffer. 

For example, for one coverslip with one probe set using the B5 initiator, combine:

HCR amplification buffer: 294 µL 
Hairpin 1 (e.g. B5H1): 3 µL 
Hairpin 2 (e.g. B5H2): 3 µL 

Briefly vortex to mix. 

 

SECTION: Probe washes and Hybridization chain reaction (HCR) amplification
38. Aspirate old amplification buffer from samples, then add 300 µL of amplification solution. Incubate 60 min to 180 min at Room temperature and in the dark.

 

SECTION: Probe washes and Hybridization chain reaction (HCR) amplification
39. Following HCR amplification, remove unassembled hairpins by washing 4 times in wash buffer (5x SSC, with 0.1% Tween-20). Perform 1 brief rinse with 1 mL of buffer, followed by three longer washes with 1 mL  at 37 Room temperature for 10 min each. 
SECTION: Probe washes and Hybridization chain reaction (HCR) amplification
40. (Optional) Label cell cytoplasm with a fluorophore-conjugated polyT probe. For each coverslip, prepare 1 mL of a 100 nM polyT probe dilution in wash buffer (5x SSC, with 0.1% Tween-20). Incubate samples in polyT probe for 60 min at Room temperature . 

Following incubation, wash samples 4 times in wash buffer (5x SSC, with 0.1% Tween-20). Perform 1 brief rinse with 1 mL of buffer, followed by three longer washes with 1 mL  at 37 Room temperature for 10 min each. 

SECTION: Probe washes and Hybridization chain reaction (HCR) amplification
42. (Optional) Label nucleus with Hoescht 33342. For each coverslip, prepare 1 mL of 1x PBS with 1/10000 Hoechst 33342. Incubate samples in Hoechst solution for 15 min at Room temperature .

Following incubation, wash samples 3 times with 1 mL of 1x PBS at 37 Room temperature for 5 min each. 

 

SECTION: Probe washes and Hybridization chain reaction (HCR) amplification
43. Mount coverslips. You will need a Kimwipe, a pair of clean forceps (suitable for lifting and manipulating coverslips), a 23g needle with the tip bent (to facilitate lifting of coverslip from the well), and a 15 mL tube with 15 mL of UltraPure water, a suitable mounting medium, and glass slides.

For each coverslip:
Place a 20 µL droplet of mounting medium on the surface of a clean glass slide. 
Using a hooked needle and forceps, lift coverslip out of the well, dip briefly into UltraPure water, then touch side of coverslip to a Kimwipe to wick away excess water. 
Carefully lower coverslip cell side-down onto the droplet of mounting medium, being careful to prevent bubbles

Once all coverslips are mounted, gently press down on each coverslip to push out excess mounting media. Store in the dark at Room temperature for at least    until mounting media cures. Once cured, excess Prolong Diamond can be easily removed by running slides under a gentle stream of Millipore water, being careful to not displace coverslips. 

Samples are now ready to image. 

 


SECTION: Probe washes and Hybridization chain reaction (HCR) amplification
41. (Optional) Label proteins with immunofluorescence staining. 

We expect that most standard immunofluorescence protocols can be used here. Below is the protocol we used for labeling AAV capsid proteins with immunofluorescence using a fluorophore-conjugated primary antibody. Optimal incubation conditions, dilutions, and durations, will need to be determined empirically for each protocol and antibody.

Block samples in 1 mL of BlockAid blocking buffer with 0.1% Triton X-100, for 60 min at Room temperature 
Incubate samples with 1:100 dilution of primary antibody in BlockAid blocking buffer with 0.1% Triton X-100, 4 °C for   .
Wash samples 3 times in 1x PBS-Tx, for 15 min each at Room temperature 

 

SECTION: Reagent set up
11. (Optional) Prepare 1x PBS-Tx, by combining:

10x PBS: 50 mL 
10% Triton X-100: 5 mL 
UltraPure water: 445 mL