TITLE: Protocol for in situ sequencing (ISS) in mouse skeletal muscle
AUTHORS: Ines Boehm

[DESCRIPTION]
Protocol used to perform in situ sequencing (ISS) on mouse skeletal muscle.

[STEPS]
SECTION: Sectioning
3. Clean the cryostat with 70% EtOH. Clean the block with RNA-Zap, 70%EtOH and dry afterwards
SECTION: Sectioning
4. Put the number of blades needed (as many as different tissues are being sectioned) on top of the block and turn on UV-lamp. Keep brushes and other tools that will be used also in the cryostat.

Careful! Do NOT put tissue in the cryostat at this time!
SECTION: Sectioning
6. Tissue is cryosectioned at 10µm thickness and collected on SuperFrist Plus adhesion slides and can be stored at -80°C until further use.
SECTION: Sectioning
7. Take slides with sections from -80°C and leave at room temperature (RT) for 5min to air dry (helps with tissue adherence).
SECTION: Sectioning
8. (Fixation) Add 500µL 4% PFA in 1XPBS at RT for 30 min (under the hood). From here on slides can be fully submerged in solutions (10X Genomics slide containers are very convenient).

 

SECTION: Sectioning
5. After UV-cleaning, add tissue to the cryostat, and let the tissue equilibrate to the cryostat temperature.
SECTION: Sectioning
9. Discard the solution and wash 3X with 0.5% PBST (1XPBS 0.5% Tween-20)
SECTION: Sectioning
10. (Permeabilization) Permeabilize tissue with 0.1M HCl in RNase-free H2O at RT for 5min
SECTION: Sectioning
11. Discard the solution and wash 2x with 1XPBS.
The washing itself is important, it is less important to wash it for a particular time.
SECTION: Sectioning
12. Submerge slides in 50% Ethanol for 5min.
SECTION: Sectioning
13. Submerge slides in 70% Ethanol for 5min.
SECTION: Sectioning
14. Submerge slides in 100% Ethanol for 3h at -20°C (here one can incubate over-night).
SECTION: Sectioning
15. (Protease) Digest tissue with Protease III (RNAscope kit) for 30min at RT.
SECTION: Sectioning
16. Wash slides 2x with 1XPBS.
SECTION: Sectioning
17. Wipe surface around tissue section dry, apply SecureSeal hybridization chamber.
SECTION: Sectioning
18. Add 100µL PBS-Tween (0.5% Tween-20), incubate for 1 min at RT (NOTE: PBST makes it easier for liquid to spread within hybridization chamber).
SECTION: Sectioning
19. Remove the solution from the chamber and wash with 1XPBS. Leave the sections in 1XPBS until the next step.
SECTION: Hybridisation of Padlock Probes (over-night)
20.  Prepare the following mix (100µL for one slide):
Heat the probes to 92°C for 2min (maximum) and cool on ice, spin down
 
 
SECTION: Hybridisation of Padlock Probes (over-night)
21. Remove 1XPBS from the chambers
SECTION: Hybridisation of Padlock Probes (over-night)
22. Pipette the mix into the chamber and seal using the SealTabs which come with SecureSeal chambers
SECTION: Hybridisation of Padlock Probes (over-night)
23. Incubate overnight at 37°C in RNAScope oven
SECTION: Ligation of the padlock probes
24. Remove the hybridisation mix from the chamber and wash with 1xPBST and then 1xPBS - the chamber might start to look "dirty" on the outside–this is okay, there is no need to wipe it down all the time.
SECTION: Ligation of the padlock probes
25. Prepare the following mix (100µL for one slide):
 

SECTION: Ligation of the padlock probes
26. Remove 1XPBS from the chambers
SECTION: Ligation of the padlock probes
27. Pipette the mix into the chamber and seal using the SealTabs which come with SecureSeal chambers
SECTION: Ligation of the padlock probes
28. Incubate at 37°C in RNAscope oven for 2h
SECTION: Rolling Circle Amplification
29. Turn temperature of oven down to 30°C.
Remove the ligation mix from the chamber and wash 1 x PBST and 1 x PBS 
SECTION: Rolling Circle Amplification
30. Prepare the following mix:
 

SECTION: Rolling Circle Amplification
31. Remove 1XPBS from the chambers
SECTION: Rolling Circle Amplification
32. Pipette the mix into the chamber and seal using the SealTabs which come with SecureSeal chambers
SECTION: Rolling Circle Amplification
33. Incubate at 30°C in RNAscope oven for 6h.
SECTION: Round 1:
38. After preparing the mix, filter through a 0.2um filter (you get ~half the volume).
add following mix (readout + imaging oligo hybridisation mix)

 

SECTION: Round 1:
35. Heat Readout oligos and fluorescent adaptors at 92C for 2 min (not longer). Place back on ice. Spin down.
SECTION: Rolling Circle Amplification
34. In the meantime thaw readout and imaging oligos and prepare Round 1 for readout + imaging oligo hybridization.
SECTION: Round 1:
36. Remove the Rolling circle amplification mix from the chamber and wash 2 times with 1xPBS.
Heat RNAscope oven back up to 37°C.
SECTION: Round 1:
37. At this point, the chambers can be removed and a barrier can be drawn with a pap pen. This allows for the use of less volume.
SECTION: Round 1:
39. Apply readout hybridisation mix to the chamber and incubate at 37°C in RNAscope oven for 30min.
SECTION: Round 1:
40. Discard the mix and wash 2x with 2xSSC + 0.03% LDS (Lithium Dodecyl Sulfate)
SECTION: Round 1:
41. Add TrueVIEW and incubate for ~1min (NOT longer than 2min!!!!) - do not use detergent after this step for wash steps as it washes out TrueVIEW
SECTION: Round 1:
42. Wash 2x with 1xPBS
SECTION: Round 1:
43. Add DAPI (1:1000 1XPBS) for nuclei stain and incubate for 10min at RT
SECTION: Round 1:
44. Wash 2x with 1xPBS
SECTION: Round 1:
45. Add ~20µL of mounting media on top of the tissue - ProLong Diamond, coverslip and store in the dark at 4°C until imaging the next day.
SECTION: Round 2:
46. Decoverslip in tube of 1XPBS - wait for coverslip to fall off - this could be overnight if slide had been mounted more than a day ago.
Decoverslipping in 2xSSC + 0.03% LDS also works.
SECTION: Probe Design
1. Padlock probes can be designed in a Jupyter Notebook using the following directions:
https://github.com/Moldia/Lee_2023/tree/main/PLP_directRNA_design

Padlock probes can also be designed using dicey from the command line:
https://github.com/gear-genomics/dicey
or their website user interface (not so suitable for large scale input)
https://www.gear-genomics.com/padlock/

Additionally, complementary oligo sequences to design FISH probes or padlock probes can be taken from https://paintshop.io/ - the padlock probes have to be assembled manually in that case.
SECTION: Round 2:
47. Strip imaging oligos with 100% Formamide 2x for 2min.
Formamide is toxic and has to be discared separately. Work under the hood!
SECTION: Round 2:
48. Wash 2x with 2xSSC + 0.03% LDS
SECTION: Probe Design
2. Ordering Padlock Probes
Publications mention the probes had been ordered as Custom RNA oligos, we could only order as Custom DNA Ultramer from Switzerland (this might be Switzerland specific?)
Go to Custom DNA oligos
Ultramer DNA Oligo 4nmol scale is enough
Add /5Phos/ to 5`-
Add r in front of last base end 3` (we need an RNA base there)
If it’s a T we will make a U out of it (there are no Ts in RNA...)
Standard Desalting (there is no other choice)
when ordering many targets I asked to have each padlock probe phosphorylated, and subsequently all padlock probes corresponding to one target pooled and resuspended in IDTE at 200µM

Preferably rA and rC for better fidelity of Ligase - if you have a choice of sequences select ones ending in rA or rC
https://doi.org/10.1261/rna.066753.118 
SECTION: Round 2:
49. Prepare the mix for R2 as per previous table (according to correct barcode combinations).
SECTION: Round 2:
50. apply readout hybridisation mix to the chamber and incubate at 37°C in RNAscope oven for 30min - make a humidity chamber
SECTION: Round 2:
51. Discard the mix and wash 2x with 2xSSC + 0.03%LDS
SECTION: Round 2:
52. Add TrueVIEW and incubate for ~1min (NOT longer than 2min!!!!) - do not use detergent after this step for wash steps as it washes out TrueVIEW
SECTION: Round 2:
53. Wash 2x with 1xPBS
SECTION: Round 2:
54. Add DAPI (1:1000 1XPBS) for nuclei stain and incubate for 10min at RT
SECTION: Round 2:
55. Wash 2x with 1xPBS
SECTION: Round 2:
56. Add ~20µL of mounting media on top of the tissue - ProLong Diamond, coverslip and store in the dark at 4°C until imaging the next.
SECTION: Round 3 &amp; 4 + Membrane Stain:
57. Proceed the same for Round 3 and Round 4.
SECTION: Round 3 &amp; 4 + Membrane Stain:
58. After hybridisation of Round 4 stain for cell-membrane markers. We stain for Laminin.
SECTION: Round 3 &amp; 4 + Membrane Stain:
59. Remove round 4 hybridisation mix and wash 2x with 2xSSC + 0.03%LDS
SECTION: Round 3 &amp; 4 + Membrane Stain:
60. Wash 2x with 1XPBS
SECTION: Round 3 &amp; 4 + Membrane Stain:
61. Block section with 0.4% Triton-X, 3%BSA in 1XPBS for 30min at RT
SECTION: Round 3 &amp; 4 + Membrane Stain:
62. Incubate in primary antibody rabbit anti-laminin (1:100 in 3% BSA in 1XPBS) for 1h at RT.
SECTION: Round 3 &amp; 4 + Membrane Stain:
63. Wash 4x with 1XPBS
SECTION: Round 3 &amp; 4 + Membrane Stain:
64. Incubate in secondary AB mix (Dk anti-Rb 488 1:100 in 3%BSA in 1XPB) for 1h at RT 
SECTION: Round 3 &amp; 4 + Membrane Stain:
65. Wash 4x with 1XPBS
SECTION: Round 3 &amp; 4 + Membrane Stain:
66. Add TrueVIEW and incubate for ~1min (NOT longer than 2min!!!!) - do not use detergent after this step for wash steps as it washes out TrueVIEW
SECTION: Round 3 &amp; 4 + Membrane Stain:
67. Wash 2x with 1xPBS
SECTION: Round 3 &amp; 4 + Membrane Stain:
68. Add DAPI (1:1000 1XPBS) for nuclei stain and incubate for 10min at RT
SECTION: Round 3 &amp; 4 + Membrane Stain:
69. Wash 2x with 1xPBS
SECTION: Round 3 &amp; 4 + Membrane Stain:
70. Add ~20µL of mounting media on top of the tissue - ProLong Diamond, coverslip and store in the dark at 4°C until imaging the next.