TITLE: HybISS: Hybridization-based In Situ Sequencing
AUTHORS: Daniel Gyllborg, Mats Nilsson

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Protocol for multiplexed in situ sequencing in tissue sections as an image-based spatial transcriptomic method. This is the second iteration of In Situ Sequencing (HybISS: Hybridization based In Situ Sequencing) based on the principles published in Ke et al. Nature Methods, 2013 and more recently Qian et al. Nature Methods, 2019. Here we present a modified method in detection chemistry using sequencing by hybridization approach for combinatorial decoding. This results in a more robust method for detecting larger probe panels for a more high-throughput in situ sequencing method.</div></div>

[STEPS]
?. [Tissue Sample Preparation: Mouse Section]
!!! Note: For human sections, proceed to Step 4 !!!Fresh frozen tissue samples are embedded in OCT (optimal cutting temperature) compound and stored at -80°C until cryosectioning. Tissue is cryosectioned at 10 μm thickness and collected on SuperFrost Plus adhesion slides and can be stored at -80°C until used for experiment.
Cryosections thickness of 5-20 µm have been tested. We typically cryosection at a thickness of 10 µm for optimal results.
?. [Tissue Sample Preparation: Mouse Section]
Slides with sections are taken from -80°C and left at room temperature (RT) for 3-5 min to air dry.
[Thaw at RT]
?. [Tissue Sample Preparation: Mouse/Human Section]
After permeabilization, tissue sections are washed with PBS two times.
?. [Tissue Sample Preparation: Mouse/Human Section]
The tissue is permeabilized with 0.1 M HCl (in H2O) at RT for 5 min.
This can be performed by submerging glass slide in ~20 ml solution of 0.1 M HCl, in a jar or container that can hold slides.
[HCl permeabilization]
Safety precaution: Hydrochloric acid
Permeabilization of tissue needs to be optimized. Additional pre-treatments are possible such as incubation with Pepsin. Permeabilization should be optimized depending on the source of the tissue.
?. [Tissue Sample Preparation: Mouse/Human Section]
Sections are then ethanol dehydrated in order to aid thin the adhesion of SecureSeal chambers.Submerge sections/slides in 70% Ethanol for 1min.Transfer and submerge in ~100% Ethanol for 1min.Remove slide and let air dry.Note that it is also possible to mount SecureSeal chambers without dehydrating slides, you just have to ensure a dry surface area around tissue for proper adhesion of the chamber.
?. [Tissue Sample Preparation: Mouse/Human Section]
With a dry surface around tissue section, apply appropriate size SecureSeal hybridization chambers over tissue.
SecureSeal hybridization chambers come in different sizes, shapes, and depths. Small chambers have a volume of ~50 µl (round, 9 mm diameter, and 0.8 mm deep: enough for half a coronal section of a mouse brain). For larger tissue specimens, larger chambers and shapes can be used and consequently volumes in protocol should be adjusted.Chambers have to be mounted on dry surfaces for a proper seal to prevent reagent content loss during incubations.
?. [Tissue Sample Preparation: Human Section]
Fresh frozen tissue samples are embedded in OCT (optimal cutting temperature) compound and stored at -80°C until cryosectioning. Tissue is cryosectioned at 10 μm thickness and collected on SuperFrost Plus adhesion slides and can be stored at -80°C until used for experiment.
Extra precautions should be taken with tissue of human origin. Obey local guidelines/rules and take appropriate pecautionary measures when handling tissue, especially fresh tissue to avoid any risks.
?. [Tissue Sample Preparation: Human Section]
Slides with sections are taken from -80°C and left at room temperature (RT) for 3-5 min to air dry.
[Thaw at RT]
?. [Tissue Sample Preparation: Mouse Section]
Fixation performed with freshly prepared 3% formaldehyde in PBS for 5 min at RT.Formaldehyde solution is applied directly on top of the section on flat laying slide.Proceed to Step 7.
[Tissue fixation]
Safety precaution: Formaldehyde
?. [Tissue Sample Preparation: Human Section]
Fixation performed with freshly prepared 3% formaldehyde in PBS for 30 min at RT.Formaldehyde solution is applied directly on top of the section on flat laying slide.
[Tissue fixation]
Safety precaution: Formaldehyde
?. [Tissue Sample Preparation: Mouse/Human Section]
Sections and chamber are washed by applying PBS-T to chamber inlet and then followed by one wash of PBS. Let PBS remain in chamber until the next steps' reagents are prepared.
Apply and remove solutions to chamber by tilting the slide/chamber slightly and pipetting into lower inlet. This will aid in preventing bubble formation within the chamber.
?. [In Situ Reverse Transcription]
Reagents for reverse transcription are combined as in the table below.  ABCD1Reagent[Stock][Final]1x (μl)2NF-H2O34.753Reverse Transcriptase Buffer10X1X54dNTPs25 mM500 μM15BSA20 μg/μl0.2 µg/µl0.56Random Decamers100 μM5 μM2.57RNase Inhibitor (Riboprotect)40 U/μl1 U/µl1.258Reverse Transcriptase (TranscriptME)200 U/μl20 U/µl59Total50
All following tables in this protocol have been calculated to a final volume of 50 μl and should be adjusted accordingly.
ABCD1Reagent[Stock][Final]1x (μl)2NF-H2O34.753Reverse Transcriptase Buffer10X1X54dNTPs25 mM500 μM15BSA20 μg/μl0.2 µg/µl0.56Random Decamers100 μM5 μM2.57RNase Inhibitor (Riboprotect)40 U/μl1 U/µl1.258Reverse Transcriptase (TranscriptME)200 U/μl20 U/µl59Total50
?. [In Situ Reverse Transcription]
PBS is removed from SecureSeal chamber and combined reagents from table above added.Chamber inlets are sealed and slide placed in a humidity chamber.Slide and humidity chamber incubated from 6 hours to overnight at 37°C.
Any container to hold slides in place, lying flat and allow moisture retention will suffice. We use small slide boxes with wet (Milli-Q water) whatman paper in bottom of container.
[Reverse transcription incubation]
?. [Padlock Probe Hybridization and Ligation]
The next day, samples are taken out of incubator.Reverse transcription reagents are carefully removed and postfixation is performed with 3% formaldehyde in PBS for 40 min at RT. Note that there is no wash step in between.
[Postfixation]
Safety precaution: Formaldehyde
?. [Padlock Probe Hybridization and Ligation]
Remove PFA and wash chambers 2 times with PBS. Let PBS remain in chamber until the next steps' reagents are prepared.
?. [Padlock Probe Hybridization and Ligation]
Combine reagents for PLP hybridizations in table below.  ABCD1Reagent[Stock][Final]1x (µl)2NF-H2O223Ligase (Tth) buffer10X1X54KCl1M50 mM2.55Formamide100%20%106Padlock Probe(s)0.5 µM10 nM each17BSA20 µg/µl0.2 µg/µl0.58Tth DNA Ligase5 U/µl0.5 U/µl59RNaseH5 U/µl0.4 U/µl410Total50
ABCD1Reagent[Stock][Final]1x (µl)2NF-H2O223Ligase (Tth) buffer10X1X54KCl1M50 mM2.55Formamide100%20%106Padlock Probe(s)0.5 µM10 nM each17BSA20 µg/µl0.2 µg/µl0.58Tth DNA Ligase5 U/µl0.5 U/µl59RNaseH5 U/µl0.4 U/µl410Total50
The amount of padlock probes will vary depending on user/experiment and initial concentration may vary. Final concentration of each padlock probe should be around 10 nM each. This may require optimization depending on the total amount of padlock probe targets.
Safety precautions: Formamide
?. [Padlock Probe Hybridization and Ligation]
Remove PBS from chambers and add combined reagents from above table to chamber.Seal chamber inlets and place slide in humidity chamber.Incubate at 37ºC for 30 min.
37 °C
[PLP incubation (1)]
?. [Padlock Probe Hybridization and Ligation]
After 30 min incubation, transfer humidity chamber with slide to 45ºC incubator to incubate for an additional 1 hour 30 min.
45 °C
[PLP incubation (2)]
?. [Rolling Circle Amplification (RCA)]
Remove reagents from chamber after incubation.Wash chamber 2 times with PBS.Let PBS remain in chamber until next reagents are combined.
?. [Rolling Circle Amplification (RCA)]
Reagents for RCA are combined as in the table below.  ABCD1Reagent[Stock][Final]1x (µl)2NF-H2O333Phi29 buffer10X1X54Glycerol50%5%55dNTPs25 mM0.25 mM0.56BSA20 µg/µl0.2 µg/µl0.57Exonuclease I20 U/µl0.4 U/µl18Phi29 polymerase10 U/µl1 U/µl59Total50
ABCD1Reagent[Stock][Final]1x (µl)2NF-H2O333Phi29 buffer10X1X54Glycerol50%5%55dNTPs25 mM0.25 mM0.56BSA20 µg/µl0.2 µg/µl0.57Exonuclease I20 U/µl0.4 U/µl18Phi29 polymerase10 U/µl1 U/µl59Total50
?. [Rolling Circle Amplification (RCA)]
PBS from chamber is removed and combined reagents from table above are applied to chamber.
?. [Tissue Sample Preparation: Mouse/Human Section]
Formaldehyde on section is discarded and tissue section is washed with PBS at RT.
To discard solutions from slides, tilt slide to allow excess liquid to pour off into disposal container. Additional liquid can be removed by tapping long edge of slide against a paper towel.For all washing steps, use a reasonable amount of washing buffer on section. For a mouse coronal section, 200 µl is sufficient.
?. [Tissue Sample Preparation: Mouse/Human Section]
Section washed two more times with PBS.
?. [Autofluorescence Quenching: Human Tissue]
Combine reagents for TrueBlack Autofluorescence Quencher (TLAQ) as per manufacture's instructions.
For human tissue or tissue that is know to give high autofluorescence, it is recommended to treat the tissue at this point with some sort of autofluorescent quencher. Here we use TrueBlack Lipofuscin Autofluorescent Quencher. Other quenchers can also be tested and optimized.If quenching is not needed, Steps #26-28 can be skipped.
Safety precaution: TLAQ
?. [Rolling Circle Amplification (RCA)]
Seal chamber inlets and place slide in humidity chamber and incubate at 37°C for up to 4 hours and then switch to 30°C overnight.
[Incubation]
[Overnight incubation]
It is also possible to place directly at 30°C overnight if there are time constraints. Exonuclease I has an optimal working temperature at 37°C and phi29 polymerase at 30°C.
?. [Rolling Circle Amplification (RCA)]
After overnight incubation, remove reagents from chamber.Wash chamber twice with PBS. Let PBS remain until next step.
?. [Autofluorescence Quenching: Human Tissue]
Remove PBS and apply TLAQ solution to chamber.Incubate at RT for 30 sec to 1 min.
[TLAQ]
Various tissue sources could require different TLAQ incubation times that would need to be tested. For brain tissue 30sec - 2min has been tested and works well. Here, TLAQ is applied after RCA and before detection oligo probe hybridization. It is also possible to perform TLAQ treatment after detection oligo hybridization.
?. [Autofluorescence Quenching: Human Tissue]
Remove TLAQ solution and wash immediately with PBS.Repeat PBS wash 2 more times (3 total).Let PBS remain until next step.
When TLAQ has been applied, detergents and ethanol will wash out TLAQ and dilute its effects on autofluoresence. It is important to avoid them from this point on. High concentrations of formamide can also bleach out effect of TLAQ. TLAQ can be reapplied again if necessary. TLAq treated sections should also be kept in the dark as much as possible as it is light sensitive.
?. [Sequence By Hybridization: Bridge-Probe and Detection Oligo]
Combine reagents for Bridge-Probe hybridization as in table below.  ABCD1Reagent[Stock][Final]1x (µl)2NF-H2O24.532X Hybridization buffer4XSSC, 40% Formamide2XSSC, 20% Formamide254Bridge-Probes10 µM0.1 µM0.55Total50
ABCD1Reagent[Stock][Final]1x (µl)2NF-H2O24.532X Hybridization buffer4XSSC, 40% Formamide2XSSC, 20% Formamide254Bridge-Probes10 µM0.1 µM0.55Total50
?. [Sequence By Hybridization: Bridge-Probe and Detection Oligo]
PBS is removed from section and is washed once with 2XSSC. Reagents are combined from table above and are applied to section.
?. [Sequence By Hybridization: Bridge-Probe and Detection Oligo]
Incubate Bridge-probe reagents for 1 hour at RT in dark, on rocker.
[Bridge-Probe incubation]
All hybridizations (Bridge-Probe and detection oligos) at room temperature are performed on a slow, 360°, low angle rotation rocker to ensure distribution of reagents evenly on tissue sections.
?. [Sequence By Hybridization: Bridge-Probe and Detection Oligo]
Discard reagents and wash 2 times with 2XSSC.Let 2XSSC remain until next step reagents are prepared.
?. [Sequence By Hybridization: Bridge-Probe and Detection Oligo]
Combine reagents for dection oligo hybridization as in table below.  ABCD1Reagent[Stock][Final]1x (µl)2NF-H2O22.7532X Hybridization buffer4XSSC, 40% Formamide2XSSC, 20% Formamide254DO_Seq_110 µM0.1 µM0.55DO_Seq_210 µM0.1 µM0.56DO_Seq_310 µM0.1 µM0.57DO_Seq_410 µM0.1 µM0.58Anchor (see note)9DAPI100 µg/µl0.5 µg/µl0.2510Total50
ABCD1Reagent[Stock][Final]1x (µl)2NF-H2O22.7532X Hybridization buffer4XSSC, 40% Formamide2XSSC, 20% Formamide254DO_Seq_110 µM0.1 µM0.55DO_Seq_210 µM0.1 µM0.56DO_Seq_310 µM0.1 µM0.57DO_Seq_410 µM0.1 µM0.58Anchor (see note)9DAPI100 µg/µl0.5 µg/µl0.2510Total50
Anchor: If anchor is used, the same final concentration as detection oligos should be used. It is possible to work without an anchor when running image analysis. Anchor imaging is typically used for spot detection and alignment across cycles.
?. [Sequence By Hybridization: Bridge-Probe and Detection Oligo]
2XSSC removed and combined reagents from table added to chamber.
?. [Sequence By Hybridization: Bridge-Probe and Detection Oligo]
Slides are incubated in dark at RT on rocker for 1 hour.
[DO incubation]
?. [Sequence By Hybridization: Bridge-Probe and Detection Oligo]
After incubation, section is washed with PBS 2 times.
?. [Sequence By Hybridization: Bridge-Probe and Detection Oligo]
Remove as much PBS as possible and then add small drop of mounting media and apply cover slip to slide section.
We use SlowFade Gold Antifade Mountant as it has shown to perform well with our conditions and also allows for removal of cover slip in future steps.10 μl of mounting media should be enough for the area of one mouse brain coronal section.
Slides at this point can be stored at +4°C with mounted coverslips in the dark for several weeks. It is recommended to image sections soon after detection oligos are added.
?. [Imaging]
Section can now be imaged.
Labs will have different microscope set ups and identical setups are not needed. Fluorophore dyes can be interchanged depending on your own microscopes capabilities. The number of detection oligo sequences presented here is 4, but this is only limited by your microscopes ability to distinguish fluorophores and can be adjusted to be more or less which would also require adjustments to your combinatorial decoding scheme.We use a Zeiss Axio Imager.Z2 epifluorescent widefield microscope. It is equipped with either a metal halide lamp or 6-channel LED light source and sCMOS camera. A good filter setup is essential to provide good wavelength seperation and minimal crosstalk between different channels. A typical imaging run includes DAPI for nuclei, 4 seperate fluorophores for the different detection oligo sequences and one channel for anchor detection.
?. [Imaging]
Imaging is set up to do repeated cycles of same region every round. Tile region outlines are set up for the first cycle and saved. After every round/cycle, slides are placed back in stage slide holder to the same position for proper alignment. This is aided with the use of an automatic stage that can calibrate to a reference point every time it is used so tile regions have the same coordinates.
?. [Stripping Bridge-Probes and Detection Oligos]
Submerge slide with coverslip in clean PBS in a coplin jar and let sit for a few minutes. Proceed to next step during this time and prepare reagents.
?. [Stripping Bridge-Probes and Detection Oligos]
Remove slide from PBS and if cover slip does not slide off by itself, either let it sit longer in PBS or carefully slide it off with the aid of forceps.Remove excess PBS from slide.
?. [Stripping Bridge-Probes and Detection Oligos]
Wash section 2 times with PBS to remove residual mounting media
?. [Repeated Cycles]
Now the Bridge-Probe:Detection Oligo has been stripped from the tissue.From this point, next cycle can be performed as before with next cycle of Bridge-probes.
If next cycle of Bridge-probes is not done immediately, section can be stored with PBS at +4°C for short-term storage. For longer-term storage, mount section with mounting media and a coverslip and store at +4°C, then remove and wash section as before to proceed with next round.
?. [Repeated Cycles]
Repeat cycle by applying Bridge-probes for next cycle.Hybridization: Steps 29-39Imaging: Steps 40-41Stripping: Steps 42-49
?. [Stripping Bridge-Probes and Detection Oligos]
Discard PBS and wash tissue section 5 times with 2XSSC
?. [Stripping Bridge-Probes and Detection Oligos]
While slide is sumberged, prepare stripping solution and humidity chamber.Combine reagents for stripping solution as in table below. Preincubate combined reagents and humidity chamber at 30°C until used in Step 47.  ABCD1Reagent[Stock][Final]1x (µl)2NF-H2O253Formamide100%65%654SSC20X2X105Total100Prepare 2XSSC washing solution. For 5 ml: 500 μl 20XSSC + 4.5 ml NF-H2O.
ABCD1Reagent[Stock][Final]1x (µl)2NF-H2O253Formamide100%65%654SSC20X2X105Total100
Safety precaution: Formamide
?. [Stripping Bridge-Probes and Detection Oligos]
Place slide in prewarmed humidity chamber. Apply prewarmed stripping solution on top of section and place in 30°C incubator for 30 min. Place any remaining stripping solution in the incubator as well.
30 °C
[Stripping incubation]
?. [Stripping Bridge-Probes and Detection Oligos]
After 30 min, remove solution from section and wash 1 time with remaining stripping solution.
?. [Stripping Bridge-Probes and Detection Oligos]
Wash 5 times with 2XSSC
?. [Sequence By Hybridization: Bridge-Probe and Detection Oligo]
After washing steps, carefully remove SecureSeal chamber.Then wash section one more time with PBS.
Remove SecureSeal chamber with the aid of forceps.
?. [Sequence By Hybridization: Bridge-Probe and Detection Oligo]
Then carefully remove as much liquid as possible from area surrounding tissue section. Use paper towel to dry area if needed. Then, with a hydrophobic pen, draw a barrier surrounding tissue section.After barrier has been drawn around tissue and dried slightly, apply additional PBS to section to prevent it from drying out and proceed to next step.
We use ImmEdge Hydrophobic Barrier PAP Pen by Vector Laboratories (Cat. No. H-4000). Other hydrophobic pens have shown to impede the in situ sequencing visualiztion. It is also possible to reapply SecureSeal hybridization chambers instead of using hydrophobic pen for subsequent hybridization steps. We find it easier to continue all steps without the use of the chamber. Note that the same volume should be used on tissue sections as what the chamber volume was.
?. [Image Analysis]
We refer you to the accompany publication and previous publications from the lab for image analysis. All tools and pipelines are available through our GitHub page.
?. [Repeated Cycles]
Repeat cycles until combinatorial barcodes can be decoded. It is also possible to re-do cycles if problems arise with some rounds.