TITLE: Home made direct RNA detection 
AUTHORS: Hower Lee, Marco Grillo, Mats Nilsson

[DESCRIPTION]
Highly multiplexed spatial profiling of mRNAs has recently gained traction as a tool to explore cellular diversity, allowing to visualize the cell composition of tissues directly in-situ.
We propose a sensitive, open-source, flexible and customisable method for the generation of in-situ expression maps of hundreds of genes. The method exploits direct ligation of padlock probes on their target mRNAs, coupled with rolling circle amplification and hybridization-based in situ combinatorial barcoding, to achieve high detection efficiency, high throughput and large multiplexing (up to few hundred genes).

[BEFORE_START]
Embed your fresh-frozen sample in a cryo embedding mold of your choice using OCT medium.
Cut sections to a thickness of 10-20 micra and attach the sections to Superfrost slides.
Store the sections at -80 until use.

[GUIDELINES]
This protocol has been thoroughly tested on a variety of fresh frozen tissue sections coming from various animal models with minimal modification. Different tissues have sometimes slightly different cell or extra-cellular matrix composition and might need specific pre-treatments to make the tissue layers accessible to reagents and enzymes.


Equipment


Hydrophobic pen
Forceps
30°C, 37°C, and 45°C incubators
Humidity chamber for slide incubation
SecureSeal hybridization chambers (Grace Bio-Labs)
Coverslips
Coplin jars or similar for washing of slides
Adhesive microscopy slides (e.g., Menzel Gläser SuperFrost Plus)
Wide-field epifluorescence microscope (6-channel)
Plate rocker


Necessary enzymes:

T4 RNA Ligase 2
Phi29 polymerase
RNAse Inhibitor


General Guideline and Controls


Enzymes and other reagents included in this protocol can be purchased from several different manufacturers (e.g. New England Biolabs or Thermo Fisher Scientific) and have performed equally well in our hands. Optimization, testing, and benchmarking should be performed whenever new reagents from different vendors are used. 
Enzymes can be produced on the cheap by your local protein production facility, if you have one nearby. The patent rights for these enzymes have expired, we provide here links to the primary sequences for your convenience.
Stock concentrations of reagents could vary depending on vendor used. Adjust tables so that final concentration of reagents is the same. Optimization might also be required.
This protocol involves RNA work and special care needs to be taken to prevent RNases. It is recommended to have designated space and equipment for RNA work and should be treated/cleaned with commercially available RNase and DNAse inactivating agents and then wiping with 100% ethanol after treatment.
Using sterile, disposable, RNase-free plasticware (pipette tips, slide boxes, tubes, and  flasks) is recommended.
PBS and water can be purchased as RNase free from numerous vendors. It is also possible to treat PBS and water with DEPC (diethyl pyrocarbonate) to make them RNase free.
Synthetic DNA targets can be used to validate specificity of padlock probes. Rolling circle amplification (RCA) can be monitored in vitro by staining rolling circle products (RCPs) with either intercalating dyes (SYBR dyes) or decorator probes and visualized under a microscope or qPCR system.
This protocol assumes correct design of padlock probes (PLPs), Bridge-probes, and detection oligos (DOs) for sequencing. See publications for further details on probe design to target genes of interest.
This protocol does not go into detail on padlock probe design and analysis of data. See publications for further detail and image analysis.


Abbreviations


PLP: padlock probe
RCA: rolling circle amplification
RCP: rolling circle product
SBH: sequence by hybridization
DO: detection oligonucleotide

[STEPS]
SECTION: Tissue preparation and fixation
1. Embed fresh-frozen tissues in OCT (optimal cutting temperature) compound and store them at -80°C until cryosectioning. 

Section the tissues at 5-20 μm thickness and collect on SuperFrost Plus adhesion slides and store at -80°C until used for experiment.
 
When working with Drosophila tissues or other very fragile material, the tissues can be fixed for 20' in 4% PFA at room temperature, washed 4 times in PBS, cryoprotected in 30% sucrose in PBS overnight, and finally embedded in OCT. This slightly reduces the detection efficiency but keeps a much better tissue morphology.
SECTION: Tissue preparation and fixation
2. Take the slides from -80°C and leave them at room temperature (RT) for 3-5 min to air dry and reach room temperature.  

SECTION: Tissue preparation and fixation
3. Fix with freshly prepared 3% formaldehyde in PBS for 5 min at RT.
Formaldehyde solution can   be applied directly on top of the section, or the slide can be submerged in a large amount of fixative. 
 


SECTION: Tissue preparation and fixation
4. Discard formaldehyde from tissue section and wash with PBS at RT 3 times

SECTION: Tissue preparation and fixation
5. Permeabilize the tissue with 0.1 M HCl (in H2O) at RT for 5 min.
This can either be done by submerging the slide in HCl or by covering the section as in step 3
  


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. In many of the cases this doesn't seem a crucial step.
SECTION: Tissue preparation and fixation
6. After permeabilisation, wash the sections with PBS two times. 
SECTION: Tissue preparation and fixation
7. Dehydrate the sections in order to aid 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.
SECTION: Tissue preparation and fixation
8. Apply SecureSeal hybridization chambers of the appropriate size over the tissue.
SECTION: Tissue preparation and fixation
9. Wash the section and the chamber by applying PBS-T to chamber inlet and then wash once with PBS. Let PBS remain in chamber until the reagents for the next steps are prepared.

The PBS-T will coat with detergent the surface of the tissue, slide and chamber, making the next steps of washing and pipetting much easier.

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. 


SECTION: Hybridisation of padlock probes
10. 
Prepare the following hybridisation mix.
 
Remove the PBS from the chamber, add the hybridisation mix and incubate at 37 degrees C overnight

 

SECTION: Probe ligation
12. Prepare the following ligation mix.
 
Remove the 2xSSC and add the ligation mix to the chamber. 
Incubate for 2 hours at 37 degrees C.
SECTION: Hybridisation of padlock probes
11. Remove the hybridisation mix.
Wash twice in 10% formamide in 2xSSC.
Wash twice in 2xSSC.
SECTION: Labeling
14. After overnight incubation, remove reagents from chamber.
Wash chamber twice with PBS. Let PBS remain until next step.
SECTION: Amplification of the padlock probes
13. Wash twice with PBS.

Prepare the following amplification mix.