Fluorescent In Situ Sequencing (FISSEQ)
Fluorescent in situ sequencing (FISSEQ) is a method of sequencing a cells's RNA while it remains in tissue or culture using next-generation sequencing.
FISSEQ combines the spatial context of RNA-FISH and the global transcriptome profiling of RNA-seq. FISSEQ preserves the tissue allowing single molecule in situ RNA localization. The foundation of the method is a novel nucleic acid sequencing library construction method that stably cross-links cDNA amplicons within biological samples. Sequencing data is then generated through an intensive interleaved microscopy and biochemistry protocol and subsequent image processing and bioinformatics. FISSEQ is compatible with diverse sample types including cell culture, tissue sections, and whole mount embryos. FISSEQ is an example of an extremely dense form of in-situ nucleic acid readout: every letter along the RNA chain is read. Thus, barcodes for FISSEQ can be packed into a short string of DNA, as short as 15-20 nucleotides long for the mouse brain or 5 nucleotides for targeted cancer gene panels
expansion microscopy
Expansion Microscopy (ExM) is a sample preparation tool for biological samples that allows investigators to identify small structures by expanding them using a polymer system.The premise is to introduce a polymer network into cellular or tissue samples, and then physically expand that polymer network using chemical reactions to increase the size of the biological structures. Among other benefits, this allows those small structures to be imaged with a wider range of microscopy techniques. It was first proposed in a 2015 article by Fei Chen, Paul W. Tillberg, and Edward Boyden.
Class Assignment
Exercise 1: Cast an expanding gel
PROTOCOL
Step 1. We prepared the Monomer Solution that can be stored at -20 for at least a month)
​
Step 2. We prepared gelation chamber, for this we needed two glass slides, two coverslips and some parafilm. Wrapped one of the slides in parafilm such that one face of the slide had a smooth flat surface one parafilm layer thick. Placed a coverslip at each end of the parafilm-wrapped slide on its smooth face. Placed this parafilm-wrapped slide with coverslips into a petri dish or any container with a lid.
Step 3. Prepared 10% (v/v) TEMED and 10% (w/v) APS.
Step 4. Prepared gelation solution, for this mixed the following 4 solutions on ice: Monomer solution, 10% TEMED (accelerator), 10% APS (initiator solution). (APS initiator solution needs to be added last to prevent premature gelation). Solutions should immediately be vortexed or pipetted up and down after mixing to ensure full mixing and the gel should then immediately be cast to prevent premature gelation.
For 50 µL gelling solution, mixed the following:
a) Monomer solution (48µl)
b) Accelerator solution (1µl): 10% TEMED (TEMED stock solution at 10%, final concentration 0.2% (w/w). (Accelerates radical generation by APS).
c) Initiator solution (1µl): APS (APS stock at 10%, final concentration 0.2% (w/w)). (This initiates the gelling process. This needs to be added last).
Step 5. Polymerize the Gel:
Pipetted 10 ul of gelation solution onto the flat surface of the parafilm-wrapped slide between the coverslips and placed the other glass slide on top so that it rests on the coverslips, leaving a small space between the two slides where the gel will polymerize.
Put a wet Kim-wipe or tissue in the container to keep the air in the container hydrated. Put the lid on the container and place it in a 37C incubator for 1 hour (or 1.5 hours at room temperature) to polymerize.
(Note that the ideal condition for polymerization is oxygen-free because oxygen exposure inhibits the polymerization process. However, only the edges of your gel will be affected by air exposure, and the middle of the gel will polymerize well.)
​
Step 6. Expand the Gel:
Pry apart the two slides using forceps, a razor blade, or some other thin edge. Put ~ 0.5 cm of deionized water in a petri dish or other container.
Wet the paintbrush with water, use it to gently peel the gel off the slide and transfer it into the container with water. Let it sit in the water for ~10 mins, then remove the water and replace it with fresh water. Repeat this wash step 2-3 more times. The gel will expand ~4-fold. Be careful not to suck the gel up into a pipettor while changing the water!
​
Step 7. Play around with the gel to see how to pick it up and transfer it to other containers. The gels are very delicate when expanded, so this takes some finesse.
Try picking up the gel by pushing it onto a coverslip using a paintbrush. If the gel is too large for this, cut it to make it smaller.
Our experiment did not present the results we expected, this could be due to a failure in some step or in the preparation of the solutions.
At the end of step 6, our gel still had a liquid consistency, so we left overnight and the next day we had the same result so we repeated the experiment.
We performed the experiment 3 times, the second and third time the gel showed a solid consistency but when it expanded in the water it dissolved.
note
