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.

Prepare two jars of DEPC-1xPBST and keep one at 4°C.

A	B
10x PBS	8
10% Tween-20	0.8
DEPC-H2O	72

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

A	B
DEPC-water	52
16% PFA	20
10X PBS	8

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 1h 0m 0s at 4°C .

1h 0m 0s

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.

0h 3m 0s

0h 3m 0s

Prepare jars of 50%, 70% and two 100% ethanol. Dehydrate tissue sections with

0h 5m 0s

0h 5m 0s

0h 5m 0s

0h 5m 0s

Take the coverslips out of the sample holder.

0h 5m 0s

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.

Permeabilize the tissue section with 0.25% Triton X-100 in DEPC-1X PBS

0h 10m 0s

A	B
10X PBS	40
DEPC-H2O	350
RNase inhibitor (enzymatics) (40U/ul)	2
10% Triton X-100	10
SUPERase In (20U/ul)	1

Wash thrice with cold PBSTR and cold DEPC-Water

200µL for 0h 3m 0s

200µL for 0h 3m 0s

1mL quick wash

Digest with 0.01% Pepsin in 0.1N HCl. Pre-warm the pepsin to 37°C before use.

100µL for0h 1m 30s at 37

A	B
1% pepsin	1
0.1N HCl	99

Wash two times with cold PBSTR200µL for 0h 3m 0s

Prepare Reverse Transcription Mix on Ice.

A	B
DEPC-H2O	88.125
Acr_dc10-Cy5_N9 (100uM)	3.75
Acr_dc7-488_dT20 (100uM)	3.75
5X SSIV Buffer	30
0.1 M DTT	7.5
10 mM dNTP	3.75
4 mM aminoallyl-dUTP	1.5
RNase Inhibitor (enzymatics, 40U/ul)	3.75
Superase In (20U/ul)	0.375
SuperScript IV Reverse Transcriptase	7.5

Incubate tissue sections with the Reverse Transcription Mix

150µL for 0h 10m 0s at 4°C then 0h 5m 0s 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.

Wash two times with cold PBSTR

200µL quick wash

200µL quick wash

Treat the sample with Acryloyl-X mix:

A	B
10X PBS	50
10mg/mL Acryloyl-X, SE in DMSO	10
ultrapure water	440

add 500µL to the sample and incubate for 0h 30m 0s at Room temperature

quick wash with PBSTR

400µL quick wash

Incubate the sample with 300µL for 0h 30m 0s at Room temperature .

Prepare Acrylamide Solution.

A	B
10X PBS	50
40% Acrylamide/Bis (37:1)	50
SUPERase-In RNase inhibitor(20U/uL)	1.25
Enzymatic RNase inhibitor	2.5
ultrapure water	400

In the mean time prepare 5% TEMED: 5µL in 95µL

Prepare 4% APS: 10mg in 250µL

RNaseZap and UV 18mm coverslips and treat them with Gel-Slick.

Prepare the polymerization mix and mix well by gently pipetting up and down.

Note : Be quick at this step

A	B
Acrylamide solution	138
4% APS	6
5% TEMED	6

Aspirate the acrylamide solution and add 30µL to the sample and immediately cover with Gel-Slick-treated coverslip for 0h 30m 0s at Room temperature in a dark Argon chamber.

Wash with 1x PBST for 3min twice

1mL for 0h 3m 0s

1mL for 0h 3m 0s

Carefully remove the Gel-Slick-treated coverslip on the samples using a needle

Wash with 1xPBST for 3min twice

1mL for 0h 3m 0s

1mL for 0h 3m 0s

Prepare RNase Digestion Mix

A	B
ultrapure water	168
10X RNase H buffer	20
RNase H (5U/uL)	10
RNase Cocktail	2

Add 200µL

Incubate at 37°C for 1h 0m 0s . Cover the silicone wells.

Wash samples with 1X PBS twice.

1mL for 0h 3m 0s

1mL for 0h 3m 0s

Prepare the padlock-probe-hybridization mix according to the table below. Preheat the probe-water mix to 85°C for 0h 3m 0s 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

A	B
ultrapure water	93.1
10X Ampligase buffer	15
padlock probes ( 22.9 ng/uL)	31.9
100nM PLP1 oligos	1.5
Ampligase (5U/uL)	10

add 150µL to tissue sections

Incubate samples in the padlock-probe-hybridization mix at 37°C for 0h 30m 0s , 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.

Wash samples with 1x PBS twice.

1mL for 0h 3m 0s

1mL for 0h 3m 0s

Prepare RCA Primer Mix

A	B
ultrapure water	119
20X SSC	20
formamide	60
100 uM rca_primer	1

add 200µL to each sample and incubate at 37°C for 1h 0m 0s

Wash samples with 2xSSC.

1mL for 0h 3m 0s

1mL for 0h 3m 0s

Prepare RCA Enzyme Mix on ice.

A	B
ultrapure water	119.25
10X Phi29 polymerase buffer	15
10mM dNTP	3.75
4mM aminoallyl-dUTP	1.5
NEB BSA (20mg/mL)	7.5
ThermoFisher Phi29 polymerase	3

Add 150µL to each sample

Incubate samples in RCA Enzyme Mix at 30°C for 7h 0m 0s

Wash samples with 1x PBS twice.

1mL for 0h 3m 0s

1mL for 0h 3m 0s

Add the crosslinking mix to crosslink rolonies with BS(PEG)9. Prepare crosslinking mix.

A	B
250mM BS(PEG)9	10
10X PBS	50
ultrapure water	440

Crosslink rolonies with BS(PEG)9

500µL for 1h 0m 0s at 4Room temperature

Wash with PBS twice

1mLquick wash

1mLquick wash

Quench unreacted crosslinker with 1M Tris, pH 8.0

1mL for 0h 30m 0s at 4Room temperature

Wash with PBS twice

1mLquick wash

1mLquick wash

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

A	B
100 uM dcProbe0_AF488	1
100 uM dcProbe0_Cy3	1
100 uM dcProbe0_ATTO647N	1
100% formamide	60
20X SSC	20
ultrapure water	117

add 200µL to each sample. Incubate for 0h 10m 0s at Room temperature

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).

1mL for 0h 2m 0s

1mL for 0h 2m 0s

Strip with 1mL for 0h 5m 0s .

Wash with 1mL twice.

Repeat the decoding imaging with the next set of decoding probes (dcProbe1, dcProbe2, dcProbe3, dcProbe4, dcProbe5).

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

add 500µL to the sample. Incubate for 0h 10m 0s at Room temperature

wash the sample with 1x PBS twice. Then, image.

1mL for 0h 2m 0s

1mL for 0h 2m 0s