FISSEQ (Fluorescent In Situ Sequencing)
The FISSEQ method involves sequencing-by-synthesis via multiple single-base-extensions. A series of base additions (e.g. C A T G C A T G...) is interrupted by scanning (for data-acquisition) and chemical treatment of slides to remove signal prior to the next extension step. We are still working out various aspects of the protocol to improve read-length, but the basic protocol is presented here.
We will assume that you are beginning with a slide that has been PCR amplified, denatured, and annealed with the appropriate sequencing primer.
Our current FISSEQ protocol proceeds as:
1. Perform polymerase trapping
2. Extend with a Cy5-SS-dNTP
3. Scan slides to acquire data on base extension
4. Cleave thiol linkage to eliminate signal
5. Alkylate free sulfhydrl
6. Scan slides to confirm efficient cleavage
7. Go to Step 2.
Individual steps 1, 2, 4 and 5 are described in more detail below. See SBE protocol for discussion of appropriate scanning instrumentation and settings.
Special Recipes for FISSEQ
The following mixes are used in the below protocols. All should be made fresh on the day of the experiment. The Iodoacetamide-Alkylation Buffer mix should be stored in the dark.
BME-Bleaching Buffer Mix...............................................200 ml 1x Bleaching buffer + 6.8 ml 2-mercaptoethanol
Iodoacetamide-Alkylation Buffer Mix.................................1300ul Alkylation buffer + 10mg iodoacetamide
Nucleotide Mixes (50 uL per slide per extension).............. .49.5 uL 1x Klenow Buffer + 0.5 uL Cy5-SS-dNTP (100 uM)
1. Using a 3-cc syringe and a 0.22 micron filter, filter ~1.5 mL of acrylamide-bis (19:1, 40%) into a 1.5 mL microcentrifuge tube.
2. Prepare fresh caging mix:
1650 uL TGT solution
1125 uL fresh, filtered 40% acrylamide solution.
3. Prepare K Mix
34 uL caging mix
1.87 uL SSB
1.65 uL Klenow enzyme (50 U/uL)
4. Prepare fresh 5% APS.
0.5 g APS →10 mL dH20
5. Prepare fresh 5% TEMED
2 uL TEMED → 38 uL dH20.
6. Prepare A-T Mix
40 uL caging mix
1 uL 5% TEMED
7. Dry edges of slide with Chemwipe.
8. Add 100 uL of caging mix to gel surface to equilibrate.
9. Incubate slide in humidity chamber for 3 minutes. A humidity chamber can be as simple as an empty pipet box filled with water at the bottom.
10. Draw off liquid with a pipetman. Spin slide in centrifuge at 500 rpm for 30" to assist drying.
11. Pipet 15 uL of K Mix onto gel. Tilt gently to spread across gel surface.
12. Incubate slide in humidity chamber for 3 minutes.
13. Add 1 uL of 5% APS to A-T mix.
14. Immedietely pipet 15 uL of A-T mix to gel surface. Tilt gently to spread across gel surface.
15. Cover gel with a 18x30, #1 cover-slip.
16. Allow slide to polymerize in argon chamber for 35 minutes.
17. Pop off coverslip with razor blade and wash slides 2 x 4' with shaking in Wash 1E.
One of our current technology development issues involves gel stability, as the gel is now substantially higher % and is therefore more prone to tearing or peeling off of the slide. It is suggested that you move immedietely to the FISSEQ extension cycling after performing polymerase trapping.
1. Equilibrate gel by dipping slide in 1x Klenow Extension Buffer
2. Add 50 uL of nucleotide mix (see recipe above) to gel surface and incubate for one minute with gentle tilting.
3. Quench extension reaction by dipping slide in Quenching Buffer for 15 seconds.
4. Wash slide 2 x 4' with shaking in Wash 1E.
Cleave Thiol Linker and Alkylate Free Sulfhydryl
1. Incubate slides in a dedicated Coplin jar containing BME-Bleaching Buffer mix (see recipe above) for 10 minutes with shaking.
2. Wash slides (7 x 30”) in Wash 1E in a separate Coplin jar. Invert capped jar while washing to insure that all BME is being washed out. It doesn't take a lot of residual BME to cleave the dithiol of the base of the next extension.
3. Apply SecureSeal chamber to each slide.
4. Add ~550 uL of Iodoacetamide-Alkylation Buffer mix into SecureSeal chamber and incubate slide in dark for 5 minutes
5. Remove Iodoacetamide-Alkylation Buffer mix with pipetman and discard in designated waste container
6. Wash with ~550 uL of straight Alkylation Buffer by pipetting in and out of SecureSeal chamber several times
7. Remove SecureSeal chamber and wash slide 2 x 4’ with shaking in Wash 1E.