In situ sequencing for RNA analysis in tissue sections V.2

Overview of the general workflow of the protocol. Depending on incubation times, the days stated are approximations.

Visualization of how PLP works and direction of synthesis and design considerations.

Equipment

• Hydrophobic pen (see Note 1)
• Forceps
• 30°C, 37°C, and 45°C incubator
• Humidity chamber for slide incubation (see Note 2)
• Secure-Seal hybridization chambers (Grace Bio-Labs) (see Note 3)
• Coverslips (see Note 4)
• Coplin jars or similar for washing of slides
• Adhesive microscopy slides (e.g., Menzel Gläser SuperFrost®).
• Wide-field epifluorescence microscope (6-channel) (see Note 5)

General Guidelines and Controls

1. This protocol has been optimized for fresh frozen mouse brain sections. However, other tissues have been shown to work robustly with this protocol as well. Optimization for specific tissues may be required such as fixation and pretreatment conditions.
2. Enzymes and other reagents included in this protocol can be purchased from several well-known vendors like NEB or Thermo Fisher Scientific and perform equally well in our hands.
3. Stock concentrations of reagents could vary depending on vendor used. Adjust tables so that final concentration of reagents is the same.
4. 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 with commercially available RNase and DNAse inactivating agents and then wiping with 100% ethanol after treatment.
5. Using sterile, disposable, RNase-free plasticware (pipette tips, slide boxes, tubes, and flasks) is recommended.
6. Synthetic DNA targets can be used to validate specificity of padlock probes.
7. 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.
8. This protocol assumes correct design of padlock probes, anchors, and base library for sequencing. See publications for further details on probe design to target genes of interest. (see Note 6, 7)
9. This protocol does not go into detail on padlock probe design and analysis of data. See publications for further detail and image analysis.

1. We use ImmEdge Hydrophobic Barrier PAP Pen by Vector Laboratories (Cat. No: H-4000). Some other hydrophobic pens have shown to impede the in situ sequencing visualization.
2. Any container to hold slides in place flat and allow moisture retention (i.e. through wet whatman paper) will suffice.
3. Secure-Seal chambers come in different sizes, shapes and depths. Small chambers support ~50 μL chambers (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 volumes in protocol should be adjusted.
4. To achieve optimal optical resolution, cover glass thickness needs to be adjusted for the microscope setup used.
5. We use a Zeiss Axioimager.Z2 Epifluorescence microscope equipped with either a metal halide lamp or 6 LED light source and a Hamamatsu CCD camera. The following filter setup provides good wavelength separation and minimal crosstalk between different channels. 38HE (Zeiss) for imaging GFP/FITC/FAM dyes; SP102v2 (Chroma) for imaging Cy3 (minimal crass talk with 38HE filter); SP103v2 (Chroma) for imaging Cy3.5/TexasRed; SP104v2 (Chroma) for imaging Cy5; 49007 (Chroma) for imaging Cy7/Alexa 7.5 dyes.
6. Oligonucleotides for padlock probes, anchors, and base libraries were ordered through Integrated DNA Technologies (IDT). Upon arrival, desalted oligonucleotides are resuspended to a 100 μM stock in TE buffer (pH 8.0, IDTE) and stored at -20°C. Sequences can be checked for secondary structure using any web-based secondary structure prediction tools (OligoAnalyzer 3.1 tool from IDT).
7. Padlock probe (PLP) design software is available such as ProbeMaker at http://probemaker.sourceforge.net/. Program allows importing single or batch cDNA targets in FASTA format for automated PLP design. User defines parameters for PLPs. Current in-house Python software package utilizes ClustalW and BLAST+ using mouse transcriptome sequences from NCBI RefSeq database.
8. Keep 0.1% (v/v) DEPC in PBS or ddH2O for at least 1 hour at 37°C (or overnight at RT), followed by autoclaving to break down DEPC residue. DEPC inhibits RNases present in water, buffers or labware.
9. Any slides that enhance adhesion of tissue sections can be used. (Menzel Gläser SuperFrost® work very well in our hands). We commonly use 10 μm thick sections and sections should not be more than 20 μm.
10. It is recommended to use freshly prepared formaldehyde solutions in DEPC-PBS. Working solutions can be prepared form either higher concentration methanol-stabilized stock solution or from paraformaldehyde powder. Aliquots can be stored at −20°C. Do not freeze and thaw.
11. Tween-20 coats the chambers, facilitates buffer exchange and prevents formation of ”dead spaces” inside the chamber. We recommend adding buffers and solutions into a chamber when slide is slightly tilted to prevent bubble formation.
12. RNaseH has the highest activity at 37°C. It degrades RNA from mRNA/cDNA heteroduplex during the first 37°C incubation. After 30 min, sample is transferred to 45°C which is the optimal temperature for the Amp ligase. Addition of formamide into the mix lowers dsDNA stability (Tm of PLP arms/cDNAduplex). This enables extension of PLP arms that strengthens probe “locking” on cDNA and gives a good balance between arms melting and specific binding.
13. The optimal temperature for phi29 polymerase is 37°C. If RCA is performed for several hours (or over-night) at 37°C, RCPs may start to fragment what could interfere with accurate signal counting. If large RCPs are desired (thick tissue sections or those with high autofluorescence), we advise doing RCA at RT over-night. Such approach will generate very large but compact RCPs.
14. A double edge razor or forceps can be used to facilitate complete removal of the Secure-Seal chamber.
15. We typically apply <10 μL of mounting medium for single, 50 μL Secure-Seal chamber. Remove excess of the medium by gently pressing the slide against a coverslip (excess of medium will be absorbed by the paper towel). Far-red dyes are more susceptible to photobleaching. SlowFade® Gold Antifade Mountant works best in our experience.
16. CellProfiler is a great, user-friendly tool to aid biologists in image processing and analysis. With respect to presented protocol, CellProfiler offers scripts for cell segmentation (definition of the nucleus and the cytoplasm), RCP segmentation, and assignment of RCPs to individual cells or fluorescence measurements. All scripts can be implemented in automated pipeline, allowing for batch image processing. An example script for cell and RCP identification is available at CellProfiler website http://www.cellprofiler.org. Briefly, gray scale TIFF images (offering highest resolution, JPEG images are processed faster and can also be used) from individual fluorescence channels are loaded into the pipeline. Cells are segmented to nuclei and cytoplasm and RCPs are identified and related to neighboring cells. Finally, number of RCPs for each cell is exported as a .csv file, which can be used for post-analysis processing.