TITLE: DART-FISH Protocol
AUTHORS: Chien-Ju Chen, Kian Kalhor, Kun Zhang

[DESCRIPTION]
In the manuscript Mapping Human Tissues with Highly Multiplexed RNA in situ Hybridization
(https://doi.org/10.1101/2023.08.16.553610), we describe a highly multiplexed in situ hybridization technique based on in situ padlock probe capture and demonstrate in applicability to different human tissue types. This protocol details the rolony generation and decoding steps of DART-FISH. 

[BEFORE_START]
Prepare fresh frozen tissue sections at 10um thickness on coverslips. Store the sections in -80C and transfer on dry ice upon the start of the protocol. 

Make sure that the padlock probes have 5' phosphate. The enzymatic production of padlock probes (accompanying protocol) leaves a 5' phosphate. If probes are individually synthesized without 5' phosphate, run T4 PNK reaction and clean up the product using Zymo ssDNA/RNA clean up columns.

[STEPS]
SECTION: Preparation
1. Wash, dry and UV the silicone isolators. UV the EasyDip jars. Move away unused stuff from the bench. Wipe the working area by 70% ethanol and RNase Zap. Set the HybEZ oven 37 °C.

SECTION: Fixation
3. Prepare 80ml of 4% formaldehyde in 1x PBS. Store at 4 °C  for use in the same day.
 


SECTION: Fixation
4. Take the tissue sections that are on 25mm*60mm coverslips out of-80 °C freezer, put them on dry ice, quickly insert them into the EasyDip slide holder, submerge the slide holder in the staining jar containing 4% PFA in PBS. Fix for 60 min  at 4 °C . 
60 min 
SECTION: Fixation
5. Remove the DEPC-PBST 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: dehydration and mounting
6. Prepare jars of 50%, 70% and two 100% ethanol. Dehydrate tissue sections 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. Attach another 20mm diameter isolator on top of the already mounted 20mm isolator to increase the volume. 
SECTION: permeabilization
9. Wash thrice with cold PBSTR and cold DEPC-Water
200 µL  for 3 min
200 µL  for 3 min 
1 mL quick wash
SECTION: reverse transcription
13. Wash two times with cold PBSTR
200 µL quick wash
200 µL quick wash
SECTION: RNase digestion
17. Prepare RNase Digestion Mix

 

Add 200 µL 
Incubate at 37 °C for 60 min . Cover the silicone wells. 
SECTION: RNase digestion
18. Wash samples with 1X PBS twice.
1 mL for 3 min 
1 mL for 3 min 
SECTION: padlock probe hybridization
19. Prepare the padlock-probe-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 padlock-probe-hybridization mix.

Note: Adjust the volume and ultrapure water and padlock probes so that the final concentration of padlock probes is at 100 nM for the brain probe set and 180 nM for the kidney probe set

padlock-probe-hybridization mix 
 

add 150 µL  to tissue sections
SECTION: padlock probe hybridization
20. Incubate samples in the padlock-probe-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 so that the tissue sections won't dry out overnight.
SECTION: RCA
22. Prepare RCA Primer Mix
 
add 200 µL  to each sample and incubate at 37 °C  for 60 min 

SECTION: RCA
23. Wash samples with 2xSSC. 
1 mL  for 3 min 
1 mL  for 3 min 
SECTION: RCA
24. Prepare RCA Enzyme Mix on ice.
 
Add 150 µL  to each sample
Incubate samples in RCA Enzyme Mix at 30 °C for 420 min 

SECTION: padlock probe hybridization
21. Wash samples with 1x PBS twice.
1 mL for 3 min 
1 mL for 3 min 
SECTION: RCA
25. Wash samples with 1x PBS twice.
1 mL for 3 min 
1 mL for 3 min 
SECTION: imaging
27. 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 10 min at Room temperature 

SECTION: imaging
28. Wash with washing buffer (10% formamide in 2X SSC, 0.1% TritonX-100) twice. Then, image sample in imaging buffer (10% formamide in 2x SSC buffer).
1 mL for  2 min 
1 mL for 2 min 

SECTION: imaging
29. Strip with 1 mL  for 5 min . 
Wash with 1 mL  twice. 
Repeat the decoding imaging with the next set of decoding probes (dcProbe1, dcProbe2, dcProbe3, dcProbe4, dcProbe5).

SECTION: pepsin digestion
11. Wash two times with cold PBSTR 200 µL  for 3 min 

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
30. After images of samples stained with dcProbe0, 1, 2, 3, 4, 5 were taken, take the nuclei staining images with Draq5 staining. 

add 500 µL  to the sample. Incubate for 10 min at Room temperature 

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

SECTION: cDNA crosslinking and gel embedding
14. Treat the sample with Acryloyl-X mix:
 
add 500 µL to the sample and incubate for 30 min at Room temperature 

quick wash with PBSTR
400 µL quick wash
SECTION: rolony crosslinking
26. Add the crosslinking mix to crosslink rolonies with BS(PEG)9. Prepare crosslinking mix. 

 

Crosslink rolonies 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: Preparation
2. Prepare two jars of DEPC-1xPBST and keep one at 4 °C.

 

SECTION: reverse transcription
12. Prepare Reverse Transcription Mix on Ice.
 
Incubate tissue sections  with the Reverse Transcription Mix
150 µL for 10 min at 4 °C then 5 min at 37 °C 
Make sure to fully cover the silicone well. If you use a coverslip, do not press on it and do not let the coverslip come in contact with the reagents inside the well. 
SECTION: permeabilization
8. Permeabilize the tissue section with 0.25% Triton X-100 in DEPC-1X PBS
10 min 
 

SECTION: cDNA crosslinking and gel embedding
15. Incubate the sample with 300 µL  for 30 min  at Room temperature .
Prepare Acrylamide Solution. 
 
In the mean time 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.

SECTION: cDNA crosslinking and gel embedding
16. Prepare the polymerization mix and mix well by gently pipetting up and down.
Note: Be quick at this step 
 
Aspirate the acrylamide solution and add 30 µL  to the sample and immediately cover with Gel-Slick-treated coverslip for 30 min  at  Room temperature  in a dark Argon chamber.

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 using a needle
 
Wash with 1xPBST for 3min twice 
1 mL for 3 min 
1 mL for 3 min