TITLE: BICCN_DART-FISH
AUTHORS: Chien-Ju Chen

[DESCRIPTION]
This protocol documents DART-FISH procedures used to generate spatial transcriptomic data from human brain section for BICCN.

[STEPS]
SECTION: preparation
2. Wash, dry and UV the silicone isolators. UV the EasyDip jars. Move away unused stuff from the bench and clean and undust the working area. Set the HybEZ oven 37C.
SECTION: permeabilization
3. Prepare 75ml of 4% formaldehyde in 1x PBS. Requires two vials of 16% formaldehyde. 
 

SECTION: permeabilization
4. Take the sections that are on 25mm*60mm coverslips out of -80C on dry ice, insert them into the slide holder, submerge the slide holder in the staining jar containing 4% PFA in PBS. Fix for 60mins at 4C. 
60 min 
SECTION: permeabilization
5. Remove the DEPC-PSBT jar and the 4% PFA jar containing the samples from 4C. Insert the sample holder into the cold 1x PBST jar. Incubate for 3 minutes. Then insert the sample holder in the room temperature 1x PBST jar.  Incubate for 3 minutes.
3 min 
3 min 
SECTION: preparation
1. Make sure that the padlock probes have 5' phosphate. In case they do not, run T4 PNK reaction and clean up the product using Zymo ssDNA/RNA clean up columns.
SECTION: dehydration and mounting
6. Prepare jars of 50%, 70% and two 100% ethanol. Dehydrate Section with
5 min 
5 min 
5 min
5 min 
SECTION: dehydration and mounting
7. Take the coverslips out of the sample holder.
 5 min 
In the meantime, put 20mm diameter Press-To-Seal silicone isolators on a kipwipe on a flat surface. Carefully put the coverslips on silicone isolators, with the tissue sample in the hole. Gently press on the back of the coverslip to seal completely. You may want to stack a 20mm diameter isolator on top of the already mounted 20mm isolator to increase the volume. 
SECTION: permeabilization
8. Permeabilize sample with 0.25% Triton X-100 in DEPC-1X PBS for 10 min 
875 µL 100 µL 25 µL 

 

SECTION: permeabilization
9. Wash thrice with DEPC-1X PBS and DEPC-Water
1 mL  quick wash
1 mL quick wash
1 mL quick wash
SECTION: reverse transcription
12. Prepare Reverse Transcription Mix on Ice.
 
Incubate human brain sections  with the Reverse Transcription Mix
150 µL for 10 min at 4 °C then 5 min at 37 °C 
SECTION: reverse transcription
13. Wash with 1X PBS twice. 
1 mLquick wash
1 mLquick wash
SECTION: cDNA crosslinking
14. Add the crosslinking mix to crosslink cDNAs with BS(PEG)9. Prepare 5mM BS(PEG)9 in PBS. 

 
Crosslink cDNA with BS(PEG)9
500 µL for 60 min at 4 Room temperature

Wash with PBS twice
1 mLquick wash
1 mLquick wash

Quench unreacted crosslinker with 1M Tris, pH 8.0
1 mL for 30 min at 4 Room temperature

Wash with PBS twice
1 mLquick wash
1 mLquick wash
SECTION: RNase digestion
15. Prepare RNase Digestion Mix

 

Add 200 µL 
Incubate at 37 °C for 60 min . Cover the silicone wells. 
SECTION: RNase digestion
16. Wash samples with 1X PBS twice.
1 mLquick wash
1 mLquick wash
SECTION: padlock probe hybridization
17. Prepare the 484-gene-Hybridization Mix according to the table below. Preheat the probe-water mix to 85 °C for 3 min and immediately move them to a cold block or on ice. Then complete the Hybridization Mix.

484-gene-Hybridization Mix
 



add 150 µL  to human section

SECTION: padlock probe hybridization
18. Incubate samples in the Hybridization Mix at 37 °C for 30 min , then at 55 °C    .
For the overnight incubation, first set the Ez hyb oven to 60 °C and then change it to 55 °C as you put the samples in. Cover the sample well.
SECTION: RCA
20. Prepare RCA Primer Mix
 
add 200 µL  to each sample and incubate at 37 °C  for 60 min 

SECTION: RCA
21. Wash samples with 2xSSC. 
1 mL quick wash
1 mL quick wash
SECTION: RCA
22. Prepare RCA Enzyme Mix on ice.
 
Add 150 µL  to each sample
Incubate samples in RCA Enzyme Mix at 30 °C for 300 min 

SECTION: padlock probe hybridization
19. Wash samples with 1x PBS twice.
1 mLquick wash
1 mLquick wash
SECTION: RCA
23. Wash samples with 1x PBS twice.
1 mLquick wash 
1 mLquick wash 
SECTION: rolony crosslinking
24. Crosslink with Acryloyl-X Mix and embed the sample in gel. 

Prepare Acryloyl-X Mix
 
add 500 µL to the sample and incubate for 30 min at Room temperature 

quick wash with 1xPBS.
1 mL quick wash

Incubate the sample with 300 µL  for 30 min  at Room temperature .
Prepare Acrylamide Solution.
 
Aspirate the Acrylamide Solution. Do not wash the samples!!!

Prepare Polymerization Mix.
Prepare 5% TEMED: 5 µL in 95 µL 
Prepare 4% APS: 10 mg in 250 µL 
RNaseZap and UV 18mm coverslips and treat them with Gel-Slick.

 

Add 30 µL  to the sample and cover with Gel-Slick-treated coverslip for 30 min  at  Room temperature  in an Argon tank.

wash with 1x PBST for 3min twice
1 mL for 3 min 
1 mL for 3 min 

Carefully remove the Gel-Slick-treated coverslip on the samples

wash with 1xPBST for 3min twice 
 1 mL for 3 min 
1 mL for 3 min 
SECTION: imaging
25. stain the sample with Probe Hybridization Mix with decoding probes (Take dcProbe0_AF488, dcProbe0_Cy3, dcProbe0_ATTO647N probes as an example).

Prepare Probe Hybridization Mix
 

add 200 µL  to each sample. Incubate for 8 min at Room temperature 

SECTION: imaging
26. Wash with10% formamide in 2X SSC twice. Then, image.
1 mL for  3 min 
1 mL for 3 min 

SECTION: imaging
27. Strip with 1 mL  for 5 min . 
Wash with 1 mL  once. 
Repeat the decoding imaging with the next decoding probes (dcProbe1, dcProbe2, dcProbe3, dcProbe4, dcProbe5, dcProbe6, and dcProbe7).

SECTION: pepsin digestion
11. Wash twice with DEPC-1X PBS
1 mL quick wash
1 mL quick wash
SECTION: pepsin digestion
10. Digest with 0.01% Pepsin in 0.1N HCl. Pre-warm the pepsin to 37 °C before use.
100 µL for90 s at 37 10 
 

SECTION: imaging
28. After images of samples stained with dcProbe0, 1, 2, 3, 4, 5, 6, 7 were taken, take the nuclei staining images with Draq5 staining. 

add 500 µL  to the sample. Incubate for 5 min at Room temperature 

wash the sample with 1x PBS twice. Then, image.
1 mL  for 2 min 
1 mL  for 2 min