Mar 25, 2024

Public workspace2. PERFF-seq: HCR Flow-FISH and Polymer Disassembly Protocol 

DOI
https://dx.doi.org/10.17504/protocols.io.8epv5x8r4g1b/v1
2. PERFF-seq: HCR Flow-FISH and Polymer Disassembly Protocol
  • Tsion Abay1,2,
  • Robert Stickels1,2,
  • Meril Takizawa3,
  • Ronan Chaligne3,
  • Caleb Lareau4
  • 1Gladstone-UCSF Institute of Genomic Immunology, San Francisco, CA, USA;
  • 2Department of Pathology, Stanford University, Stanford CA, USA;
  • 3Single-cell Analytics Innovation Lab, Memorial Sloan Kettering Cancer Center, New York, NY, USA;
  • 4Computational and Systems Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA
Tsion Abay
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DOI: https://dx.doi.org/10.17504/protocols.io.8epv5x8r4g1b/v1
Protocol Citation: Tsion Abay, Robert Stickels, Meril Takizawa, Ronan Chaligne, Caleb Lareau 2024. 2. PERFF-seq: HCR Flow-FISH and Polymer Disassembly Protocol . protocols.io https://dx.doi.org/10.17504/protocols.io.8epv5x8r4g1b/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: February 04, 2024
Last Modified: March 25, 2024
Protocol Integer ID: 94653
Keywords: PERFF-seq, enrichment, HCR Flow-FISH, performing hcr flow, hcr flow, nuclei enrichment via fac, polymer disassembly protocol, nuclei preparation, permeabilized cell, tissue derived nuclei, hcr, nuclei enrichment, polymer disassembly, nuclei, cell
Abstract
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.
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.
Materials
  1. Hybridization Buffer (Molecular Instruments)
  2. Wash Buffer (Molecular Instruments)
  3. Amplification Buffer (Molecular Instruments)
  4. Probes and Complementary Hairpins (Molecular Instruments)
  5. SSC-T: 5X SSC, 0.1% Tween-20
  6. Sorting & Collection Buffer: 1x PBS + 5% BSA(Gibco Catalog No: 15260037) + 0.133 U/uL RNase Inhibitor (Sigma Aldrich Catalog No: 15260037)
  7. dsDNAse Thermofisher Lot No: EN0771
  8. Storage Buffer: Water + 1x Quenching Buffer + 0.1 Enhancer (warmed for 10 minutes at 65C)
  9. Long Term Storage Buffer: Water + 1x Quenching Buffer + 10% Glycerol (Millipore Sigma Catalog No: G5516-100ML) + 0.1 Enhancer (warmed for 10 minutes at 65C)
  10. Post Storage Processing buffer: 0.5x PBS + 0.02% BSA and 0.2U/uL RNase Inhibitor
  11. Thermomixer with Heated Lid or a Rotating Hybridization Oven
  12. Swinging Bucket Rotor

Troubleshooting
Safety warnings
Hybridization and Wash buffers contain formamide which is a hazardous material.
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)


Detection Stage
After performing cell/nuclei preparation, pre-warm hybridization buffer and thermomixer with a heated lid to Temperature37 °C .

Toxic
Add 400uL of pre-warmed hybridization buffer per 1 million (1M) cells/nuclei.
For cells, incubate in a thermomixer at Temperature37 °C for 30 minutes (300rpm shaking) or a rotating hybridization oven.
For nuclei, incubate samples at Temperature37 °C with no shaking.

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
After incubating, add 100uL of the prepared probe mix into each sample for a final probe concentration of 32nM.
Incubate for 16-24 hours.
For cells, incubate in a thermomixer at Temperature37 °C (300rpm).
For nuclei, incubate samples at Temperature37 °C with no shaking.
Overnight
After overnight incubation, add 500uL of SSC-T into samples and centrifuge at 850xg for 10 minutes.
Gently remove supernatant (Leaving few uLs behind to make sure not to perturb fragile pellet).
Add 500uL of wash buffer per 1M cells/nuclei and incubate at Temperature37 °C for 10 minutes.

Centrifuge at 850xg for 5 minutes and remove supernatant.
Repeat steps 8 and 9, 3 more times for a total of 4 washes.
Add 500uL of SSC-T per 1M cells/nuclei and incubate for 5 minutes at room temperature to remove formamide from sample.
Centrifuge at 850xg for 5 minutes and remove supernatant.
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 Temperature4 °C .
Pause
Amplification Stage
Add 150uL of amplification buffer per 1M cells/nuclei and incubate for 30 minutes at room temperature.
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.
Heat shock at Temperature95 °C for 90 seconds using a thermocycler.

Remove strip-tubes from thermocycler and incubate at room temperature in the dark for 30 minutes.

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

Add 100uL of hairpin solution to each sample for a final hairpin concentration of 60nM and incubate at least 4 hours or overnight at Temperature25 °C or TemperatureRoom temperature .

For cells, 300rpm shaking
For nuclei, no shaking
Overnight
To wash unbound hairpins, add 500uL of SSC-T and centrifuge at 850xg for 5 minutes.
Repeat step 20, 4 more times for a total of 5 washes.
Sample Enrichment Using FACS
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.
Re-suspend cells/nuclei in collection buffer.
  • Ideally, cells/nuclei should be filtered through a 35um filter before sorting.
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 Temperature4 °C .

Pause
HCR Polymer Disassembly
After sorting, centrifuge cells/nuclei at 850xg for 5 minutes and gently remove supernatant.
Resuspend in 275uL of 1x DNase I buffer at TemperatureRoom 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

Add 25uL of DNase I enzyme (0.5U/ul enzyme in 1x buffer) to solution and incubate at Temperature37 °C for 2 hours.

Inactivate DNase I by incubating the sample at Temperature55 °C for 5 minutes in the presence of 10 mM DTT (final).

Centrifuge at 850xg for 5 minutes to pellet cells/nuclei.

Re-suspend in 500uL wash buffer and incubate at Temperature37 °C for 10 minutes.

Repeat steps 28 and 29 for a total of 2 washes.

Resuspend in 500uL PBS-T and incubate for 5 minutes at TemperatureRoom temperature .
Optional: Resuspend in 1x PBS buffer and analyze on flow cytometer to ensure stripping.
Expected result

Expected Result from dsDNase Stripping


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.
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 Temperature4 °C .

Pause
Protocol references
1. Reilly, S. K. et al. Direct characterization of cis-regulatory elements and functional dissection of complex genetic associations using HCR-FlowFISH. Nat. Genet. 53, 1166–1176 (2021).
2. HCR‱ RNA flow cytometry protocol for mammalian cells in suspension, Molecular Instruments.
3. Chromium Fixed RNA Profiling Protocol, 10x Genomics
4. PERFF-seq: Cell and Nuclei Preparation