Jul 11, 2024
Protocol for in situ sequencing (ISS) in mouse skeletal muscle
This protocol is a draft, published without a DOI.
- 1University of Basel
Protocol Citation: Ines Boehm 2024. Protocol for in situ sequencing (ISS) in mouse skeletal muscle. protocols.io https://protocols.io/view/protocol-for-in-situ-sequencing-iss-in-mouse-skele-dghu3t6w
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: July 01, 2024
Last Modified: July 11, 2024
Protocol Integer ID: 102676
Funders Acknowledgements:
Freenovation
Disclaimer
This protocol has been adapted from Lee et al 2023: https://doi.org/10.1101/2023.10.10.561689
Abstract
Protocol used to perform in situ sequencing (ISS) on mouse skeletal muscle.
Materials
| Item | Supplier | Catalog number | |
| OCT (Optimal Cutting Temperature) Compound | CellPath | KMA-0100-00A | |
| SuperFrost Plus Adhesion Microscope Slides Blue tab | Epredia | J1810AMNZ | |
| PFA | Electron Microscopy Sciences | 15714 | |
| 10X PBS Buffer pH 7.4 1000mL | Invitrogen, Thermo Fisher Scientific | AM9625 | |
| Nuclease-free water: UltraPure ‱ Distilled Water DNase/RNase Free 500mL | Invitrogen, Thermo Fisher Scientific | 10977-035 | |
| Absolute EtOH: ACS,ISO,Reag. Ph Eur Ethanol absolute for analysis | EMSURE | 1.00983.1000 | |
| SSC (20X), RNase-free | Invitrogen, Thermo Fisher Scientific | AM9763 | |
| Lithium Dodecyl Sulfate | Sigma Aldrich | L9781-25G | |
| TWEEN 20 | Sigma Aldrich | P1379-250ML | |
| 2.5LT Hydrochloric acid solution 1M (1N), NIST Standard solution ready to use, Eur.Ph. , USP, BP | ThermoFisher Scientific | 10487830 | |
| Vector® TrueVIEW™ Autofluorescence Quenching Kit | Adipogen LifeSciences | VC-SP-8400-KI01 | |
| SECURESEAL(TM) HYBRIDIZATION CHAMBERS, 0.8mm | SigmaAldrich | GBL621502-50EA | |
| phi29 DNA Polymerase (10 U/µL) 1000U | LifeTechnologies | EP0092 | |
| GeneAmp™ dNTP Blend (100 mM) | LifeTechnologies | N8080261 | |
| T4 RNA Ligase 2 (dsRNA Ligase) | NewEngland Biolabs | M0239L | |
| ProLong Diamond Antifade Mountant 10mL | LifeTechnologies | P36970 | |
| Murine RNase Inhibitor, 15'000 | NewEngland Biolabs | M0314L | |
| Rabbit anti-Laminin Antibody (0.5 mg/mL, affinity isolated antibody, buffered aqueous solution) | Merck | L9393-100UL | |
| Alexa Fluor® 488 AffiniPure™ Donkey Anti-Rabbit IgG (H+L) | Jackson Immuno | AB_2313584 | |
Probe Design
Probe Design
Padlock probes can be designed in a Jupyter Notebook using the following directions:
Padlock probes can also be designed using dicey from the command line:
or their website user interface (not so suitable for large scale input)
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.
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
Sectioning
Sectioning
Clean the cryostat with 70% EtOH. Clean the block with RNA-Zap, 70%EtOH and dry afterwards
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!
After UV-cleaning, add tissue to the cryostat, and let the tissue equilibrate to the cryostat temperature.
Tissue is cryosectioned at 10µm thickness and collected on SuperFrist Plus adhesion slides and can be stored at -80°C until further use.
Take slides with sections from -80°C and leave at room temperature (RT) for 5min to air dry (helps with tissue adherence).
(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).
Representative image of plastic boxes we received for Visium
experiments from 10X Genomics.
Discard the solution and wash 3X with 0.5% PBST (1XPBS 0.5% Tween-20)
(Permeabilization) Permeabilize tissue with 0.1M HCl in RNase-free H2O at RT for 5min
Discard the solution and wash 2x with 1XPBS.
The washing itself is important, it is less important to wash it for a particular time.
Submerge slides in 50% Ethanol for 5min.
Submerge slides in 70% Ethanol for 5min.
Submerge slides in 100% Ethanol for 3h at -20°C (here one can incubate over-night).
(Protease) Digest tissue with Protease III (RNAscope kit) for 30min at RT.
Wash slides 2x with 1XPBS.
Wipe surface around tissue section dry, apply SecureSeal hybridization chamber.
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).
Remove the solution from the chamber and wash with 1XPBS. Leave the sections in 1XPBS until the next step.
Hybridisation of Padlock Probes (over-night)
Hybridisation of Padlock Probes (over-night)
Prepare the following mix (100µL for one slide):
Heat the probes to 92°C for 2min (maximum) and cool on ice, spin down
| Reagents | Stock concentration | Final concentration | Volume (µL)one slide | Volume (µL) | |
| RNase-free H2O | 76.1 | 456.6 | |||
| SSC | 20x | 2x | 10 | 60 | |
| Formamide | 100% | 10% | 10 | 60 | |
| BSA | 50µg/µL | 0.2µg/µL | 0.4 | 2.4 | |
| RNase inhibitor | 40U/µL | 1U/µL | 2.5 | 15 | |
| Padlock probes | 1µM eachPool of 1µM each | 10nM each | 1 | 6 | |
| TOTAL | 100 | 600 |
Remove 1XPBS from the chambers
Pipette the mix into the chamber and seal using the SealTabs which come with SecureSeal chambers
Incubate overnight at 37°C in RNAScope oven
Ligation of the padlock probes
Ligation of the padlock probes
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.
Prepare the following mix (100µL for one slide):
| Reagents (all bought from IDT) | Stock concentration | Final concentration | Volume (µL)one slide | Volume (µL) | |
| BSA (ultrapure) | 50µg/µL | 0.2µg/µL | 0.4 | 2.4 | |
| RCA primer (adpt1 primer)(Now: SF-FWD Primer) | 100µM | 5µM | 1 | 6 | |
| RNase inhibitor | 40U/µL | 1U/µL | 1.5 | 15 | |
| RNase-free H2O | 81.1 | 486.6 | |||
| T4 Rn ligase 2 buffer | 10x | 1x | 10 | 60 | |
| T4 Rnl2 Ligase | 10U/µL | 0.5U/µL | 5 | 30 | |
| TOTAL | 100 | 600 |
Remove 1XPBS from the chambers
Pipette the mix into the chamber and seal using the SealTabs which come with SecureSeal chambers
Incubate at 37°C in RNAscope oven for 2h
Rolling Circle Amplification
Rolling Circle Amplification
Turn temperature of oven down to 30°C.
Remove the ligation mix from the chamber and wash 1 x PBST and 1 x PBS
Prepare the following mix:
| Reagents | Stock concentration | Final concentration | Volume (µL) | Volume (µL) | |
| RNase-free H2O | 68.6 | 411.6 | |||
| Phi29 buffer | 10x | 1x | 10 | 60 | |
| Glycerol | 50% | 5% | 10 | 60 | |
| dNTPs (92C 2min) | 25mM per dNTP | 0.25mM | 1 | 6 | |
| BSA | 50µg/µL | 0.2µg/µL | 0.4 | 2.4 | |
| Phi29 Polymerase | 10U/µL | 1U/µL | 10 | 60 | |
| TOTAL | 100 | 600 |
Remove 1XPBS from the chambers
Pipette the mix into the chamber and seal using the SealTabs which come with SecureSeal chambers
Incubate at 30°C in RNAscope oven for 6h.
In the meantime thaw readout and imaging oligos and prepare Round 1 for readout + imaging oligo hybridization.
Round 1:
Round 1:
Heat Readout oligos and fluorescent adaptors at 92C for 2 min (not longer). Place back on ice. Spin down.
Remove the Rolling circle amplification mix from the chamber and wash 2 times with 1xPBS.
Heat RNAscope oven back up to 37°C.
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.
After preparing the mix, filter through a 0.2um filter (you get ~half the volume).
add following mix (readout + imaging oligo hybridisation mix)
| Reagents | Stock concentration | Final concentration | Volume (µL)one slide | Volume (µL) | |
| RNase-free H2O | 34 | 192 | |||
| SSC | 20x | 2x | 10 | 60 | |
| Formamide | 100% | 20% | 20 | 120 | |
| Readout oligos (for adpt1, adpt2 & adpt3) + imaging oligos: adpt1 + adpt2 + adpt3 | 10µM | 0.1µM | 1 µL each (33 targets = 33µL total) + 3µL= 36µL | 6 each(210µL total)+ 18µL=228 | |
| TOTAL | 100 | 600 |
Apply readout hybridisation mix to the chamber and incubate at 37°C in RNAscope oven for 30min.
Discard the mix and wash 2x with 2xSSC + 0.03% LDS (Lithium Dodecyl Sulfate)
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
Wash 2x with 1xPBS
Add DAPI (1:1000 1XPBS) for nuclei stain and incubate for 10min at RT
Wash 2x with 1xPBS
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.
Round 2:
Round 2:
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.
Strip imaging oligos with 100% Formamide 2x for 2min.
Formamide is toxic and has to be discared separately. Work under the hood!
Wash 2x with 2xSSC + 0.03% LDS
Prepare the mix for R2 as per previous table (according to correct barcode combinations).
apply readout hybridisation mix to the chamber and incubate at 37°C in RNAscope oven for 30min - make a humidity chamber
Discard the mix and wash 2x with 2xSSC + 0.03%LDS
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
Wash 2x with 1xPBS
Add DAPI (1:1000 1XPBS) for nuclei stain and incubate for 10min at RT
Wash 2x with 1xPBS
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.
Round 3 & 4 + Membrane Stain:
Round 3 & 4 + Membrane Stain:
Proceed the same for Round 3 and Round 4.
After hybridisation of Round 4 stain for cell-membrane markers. We stain for Laminin.
Remove round 4 hybridisation mix and wash 2x with 2xSSC + 0.03%LDS
Wash 2x with 1XPBS
Block section with 0.4% Triton-X, 3%BSA in 1XPBS for 30min at RT
Incubate in primary antibody rabbit anti-laminin (1:100 in 3% BSA in 1XPBS) for 1h at RT.
Wash 4x with 1XPBS
Incubate in secondary AB mix (Dk anti-Rb 488 1:100 in 3%BSA in 1XPB) for 1h at RT
Wash 4x with 1XPBS
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
Wash 2x with 1xPBS
Add DAPI (1:1000 1XPBS) for nuclei stain and incubate for 10min at RT
Wash 2x with 1xPBS
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.
Protocol references
Based on the original protocol in Lee et al 2023: https://doi.org/10.1101/2023.10.10.561689