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.

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.

Prepare 75ml of 4% formaldehyde in 1x PBS. Requires two vials of 16% formaldehyde.

A	B
component	volume (mL)
DEPC-water	52
16% PFA	20
10X PBS	8

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.

1h 0m 0s

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.

0h 3m 0s

0h 3m 0s

Prepare jars of 50%, 70% and two 100% ethanol. Dehydrate Section 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. You may want to stack a 20mm diameter isolator on top of the already mounted 20mm isolator to increase the volume.

Permeabilize sample with 0.25% Triton X-100 in DEPC-1X PBS for 0h 10m 0s

875µL 100µL 25µL

A	B
component	x1 volume (uL)
DEPC-water	875
10X PBS	100
10% Triton X-100	25

Wash thrice with DEPC-1X PBS and DEPC-Water

1mL quick wash

1mL quick wash

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
component	x1 volume (uL)
1% pepsin	1
0.1N HCl	99

Wash twice with DEPC-1X PBS

1mL quick wash

1mL quick wash

Prepare Reverse Transcription Mix on Ice.

A	B
component	x1 volume (uL)
DEPC-water	90.75
5X SSIV buffer	30
10mM dNTP	3.75
100uM N5_dc10-Cy5_N9	3.75
100uM dT20_dc7-488	3.75
0.1M DTT	7.5
4mM aminoallyl-dUTP	1.5
RNase inhibitor (40U/uL)	1.5
SuperScript IV Reverse Transcriptase	7.5
total	150

Incubate human brain sections with the Reverse Transcription Mix

150µL for 0h 10m 0s at 4°C then 0h 5m 0s at 37°C

Wash with 1X PBS twice.

1mLquick wash

1mLquick wash

Add the crosslinking mix to crosslink cDNAs with BS(PEG)9. Prepare 5mM BS(PEG)9 in PBS.

A	B
component	x1 volume (uL)
250mM BS(PEG)9	10
10X PBS	50
ultrapure water	440

Crosslink cDNA 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

Prepare RNase Digestion Mix

A	B
component	x1 volume (uL)
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.

1mLquick wash

1mLquick wash

Prepare the 484-gene-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 Hybridization Mix.

484-gene-Hybridization Mix

A	B
component	x1 volume (uL)
ultrapure water	98.2
10X Ampligase buffer	15
HB_Feb2022 probes ( 27.2 ng/uL)	25.3
100nM PLP1 oligos	1.5
Ampligase (5U/uL)	10

add 150µL to human section

Incubate samples in the 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.

Wash samples with 1x PBS twice.

1mLquick wash

1mLquick wash

Prepare RCA Primer Mix

A	B
component	x1 volume (uL)
ultrapure water	119
20X SSC	20
formamide	60
100 uM FISSEQ_ppRCA primer	1

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

Wash samples with 2xSSC.

1mL quick wash

1mL quick wash

Prepare RCA Enzyme Mix on ice.

A	B
component	x1 volume (uL)
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
total	150

Add 150µL to each sample

Incubate samples in RCA Enzyme Mix at 30°C for 5h 0m 0s

Wash samples with 1x PBS twice.

1mLquick wash

1mLquick wash

Crosslink with Acryloyl-X Mix and embed the sample in gel.

Prepare Acryloyl-X Mix

A	B
Component	x1 volume (uL)
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 1xPBS.

1mL quick wash

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

Prepare Acrylamide Solution.

A	B
Component	x1 volume (uL)
10X PBS	50
40% Acrylamide/Bis (37:1)	50
ultrapure water	400

Aspirate the Acrylamide Solution. Do not wash the samples!!!

Prepare Polymerization Mix.

Prepare 5% TEMED: 5µL in 95µL

Prepare 4% APS: 10mg in 250µL

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

A	B
component	x1 volume (uL)
Acrylamide solution	138
4% APS	6
5% TEMED	6

Add 30µL to the sample and cover with Gel-Slick-treated coverslip for 0h 30m 0s at Room temperature in an Argon tank.

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

wash with 1xPBST for 3min twice

1mL for 0h 3m 0s

1mL for 0h 3m 0s

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
component	x1 volume (uL)
100 uM dcProbe0_AF488	1
100 uM dcProbe0_Cy3	1
100 uM dcProbe0_ATTO647	1
100% formamide	60
20X SSC	20
ultrapure water	117

add 200µL to each sample. Incubate for 0h 8m 0s at Room temperature

Wash with10% formamide in 2X SSC twice. Then, image.

1mL for 0h 3m 0s

1mL for 0h 3m 0s

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

Wash with 1mL once.

Repeat the decoding imaging with the next decoding probes (dcProbe1, dcProbe2, dcProbe3, dcProbe4, dcProbe5, dcProbe6, and dcProbe7).

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 500µL to the sample. Incubate for 0h 5m 0s at Room temperature

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

1mL for 0h 2m 0s

1mL for 0h 2m 0s