TITLE: 2. PERFF-seq: HCR Flow-FISH and Polymer Disassembly Protocol 
AUTHORS: Tsion Abay, Robert Stickels, Meril Takizawa, Ronan Chaligne, Caleb Lareau

[DESCRIPTION]
This protocol can be used for performing HCR Flow-FISH followed by cell/nuclei enrichment via FACS and polymer disassembly on fixed and permeabilized cells and tissue derived nuclei. This protocol should be performed after finalizing "PERFF-seq: Cell and Nuclei Preparation" for the desired sample type. 

[BEFORE_START]
Calculate the starting cell count and volume of probes and hairpins needed for your experiment by putting these factors into account:
Proportion of the specific population of interest within the total cell/nuclei population.
Final cell count should be >50,000 
     i.e specific population of interest to sort
Approximately 50% of starting cells/nuclei are lost during the wash steps of this protocol.
Unstained controls and single color controls (if multiplexing) 
 


[GUIDELINES]
Maintain RNAse free environment when preparing buffers and throughout the protocol by spraying bench with RNaseZap and using molecular grade reagents when possible. 
To increase cell recovery, opt for a swinging bucket rotor when pelleting and leave a few uL of liquid behind when aspirating buffers. 
When possible, use low-binding plasticware. 

[STEPS]
SECTION: Detection Stage
1. After performing cell/nuclei preparation, pre-warm hybridization buffer and thermomixer with a heated lid to 37 °C. 

SECTION: Detection Stage
2. Add 400uL of pre-warmed hybridization buffer per 1 million (1M) cells/nuclei.
For cells, incubate in a thermomixer at 37 °C for 30 minutes (300rpm shaking) or a rotating hybridization oven.
For nuclei, incubate samples at 37 °C with no shaking.

SECTION: Detection Stage
3. In the meantime, prepare probe solution for 1M cells/nuclei by adding 8uL of each probe set (8uL from the 1uM probe stock) and top off pre-warmed hybridization buffer to a final volume of 100uL. Increase per cell count and per probe set used. 

Example: For 5 million cells/nuclei and 2 probe sets: 
Probe solution = 80uL probes (2 probe sets * 8uL * 5 million cells) + 420uL of hybridization buffer 
SECTION: Detection Stage
4. After incubating, add 100uL of the prepared probe mix into each sample for a final probe concentration of 32nM.
SECTION: Detection Stage
5. Incubate for 16-24 hours. 
For cells, incubate in a thermomixer at 37 °C (300rpm).
For nuclei, incubate samples at 37 °C with no shaking.
SECTION: Detection Stage
6. After overnight incubation, add 500uL of SSC-T into samples and centrifuge at 850xg for 10 minutes. 
SECTION: Detection Stage
7. Gently remove supernatant (Leaving few uLs behind to make sure not to perturb fragile pellet). 
SECTION: Detection Stage
8. Add 500uL of wash buffer per 1M cells/nuclei and incubate at 37 °C for 10 minutes. 

SECTION: Detection Stage
9. Centrifuge at 850xg for 5 minutes and remove supernatant. 
SECTION: Detection Stage
10. Repeat steps 8 and 9, 3 more times for a total of 4 washes. 
SECTION: Detection Stage
11. Add 500uL of SSC-T per 1M cells/nuclei and incubate for 5 minutes at room temperature to remove formamide from sample.  
SECTION: Detection Stage
12. Centrifuge at 850xg for 5 minutes and remove supernatant. 
SECTION: Amplification Stage
14. Add 150uL of amplification buffer per 1M cells/nuclei and incubate for 30 minutes at room temperature. 
SECTION: Amplification Stage
15. In the meantime, transfer 5uL of H1 and H2 hairpins per 1M cells/nuclei for each probe set 
(i.e. 5uL from the 3uM hairpin stock). 

Example: for 5M cells/nuclei and 2 probe sets:
Transfer 25uL(5M * 5uL) of h1 and h2 hairpin for each probe set (total of 4 hairpin stocks)
H1 and H2 hairpins are kept in separate tubes at this step.
SECTION: Amplification Stage
16. Heat shock at 95 °C for 90 seconds using a thermocycler. 

SECTION: Amplification Stage
17. Remove strip-tubes from thermocycler and incubate at room temperature in the dark for 30 minutes. 

SECTION: Amplification Stage
18. After incubation, prepare hairpin solution by combining h1 and h2 and adding SSC-T for final hairpin solution of 100uL per 1M cells/nuclei. 

Example: For 5M cells/nuclei and 2 probe sets: 
Hairpin solution = 100uL of hairpins (25uL*4 hairpin stocks) + 400uL of amplification buffer 

SECTION: Amplification Stage
19. Add 100uL of hairpin solution to each sample for a final hairpin concentration of 60nM and incubate at least 4 hours or overnight at 25 °Cor Room temperature.

For cells, 300rpm shaking
For nuclei, no shaking
SECTION: Amplification Stage
20. To wash unbound hairpins, add 500uL of SSC-T and centrifuge at 850xg for 5 minutes. 
SECTION: Amplification Stage
21. Repeat step 20, 4 more times for a total of 5 washes.
SECTION: Sample Enrichment Using FACS
22. If fluorophore multiplexing is performed, compensation should be done using single color control samples and unstained cells/nuclei that went through the PERFF-seq protocol. 
SECTION: Sample Enrichment Using FACS
23. Re-suspend cells/nuclei in collection buffer.
Ideally, cells/nuclei should be filtered through a 35um filter before sorting.
SECTION: Sample Enrichment Using FACS
24. Stopping point: 
Alternatively, samples can be resuspended in storage buffer (1mL per 5 million, see Materials section or 10XG protocol for more details). 
Cells or nuclei can be stored for up to 1 week at 4 °C.

SECTION: HCR Polymer Disassembly
25. After sorting, centrifuge cells/nuclei at 850xg for 5 minutes and gently remove supernatant.
Resuspend in 275uL of 1x DNase I buffer at Room temperature for 15 minutes. 

Record the amount of cells/nuclei recovered from sorting.
30uL 10x DNase I buffer + 270uL of RNase free water --> Add 275uL to sample 

SECTION: HCR Polymer Disassembly
26. Add 25uL of DNase I enzyme (0.5U/ul enzyme in 1x buffer) to solution and incubate at 37 °C for 2 hours.  

SECTION: HCR Polymer Disassembly
27. Inactivate DNase I by incubating the sample at 55 °C for 5 minutes in the presence of 10 mM DTT (final).

SECTION: HCR Polymer Disassembly
28. Centrifuge at 850xg for 5 minutes to pellet cells/nuclei.

SECTION: HCR Polymer Disassembly
29. Re-suspend in 500uL wash buffer and incubate at 37 °C for 10 minutes. 

SECTION: HCR Polymer Disassembly
30. Repeat steps 28 and 29 for a total of 2 washes. 

SECTION: HCR Polymer Disassembly
31. Resuspend in 500uL PBS-T and incubate for 5 minutes at Room temperature. 
Optional: Resuspend in 1x PBS buffer and analyze on flow cytometer to ensure stripping. 
 

SECTION: HCR Polymer Disassembly
32. For 10XG Library Prep: 
Wash 2 times with post-storage processing buffer and proceed immediately with probe hybridization step of the 10x genomics Chromium fixed RNA profiling protocol.
 
SECTION: HCR Polymer Disassembly
33. Stopping point: 
Alternatively, samples can be resuspended in storage buffer (1mL per 5 million, see Materials section or 10XG protocol for more details). 
Cells or nuclei can be stored for up to 1 week at 4 °C.

SECTION: Detection Stage
13. Stopping point: 
Samples can be resuspended in long term storage buffer (1mL per 5 million, see Materials section or 10XG protocol for more details). 
Cells or nuclei can be stored for up to 3 weeks at 4 °C.