Mar 25, 2024

Public workspace1. PERFF-seq: Cell and Nuclei Preparation 

DOI
dx.doi.org/10.17504/protocols.io.14egn3k6ql5d/v1
1. PERFF-seq: Cell and Nuclei Preparation 
  • 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
Open access
DOI: dx.doi.org/10.17504/protocols.io.14egn3k6ql5d/v1
Protocol Citation: Tsion Abay, Robert Stickels, Meril Takizawa, Ronan Chaligne, Caleb Lareau 2024. 1. PERFF-seq: Cell and Nuclei Preparation . protocols.io https://dx.doi.org/10.17504/protocols.io.14egn3k6ql5d/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 03, 2024
Last Modified: March 25, 2024
Protocol Integer ID: 94651
Keywords: PERFF-seq, Flow-FISH, Fixation, FFPE, Fresh-Frozen Tissue, Tissue Dissociation
Abstract
This protocol can be used for preparation of Cells and Fresh-Frozen and Formalin-Fixed Paraffin-Embedded (FFPE) Tissue Derived Nuclei. This protocol should be followed by "PERFF-seq: HCR-FlowFISH and Polymer Disassembly."
Guidelines
  • Maintain an 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
FOR FRESH NUCLEI PREP
  1. Dissociation Solution (Liberase TL (Lot No: 5401020001) + RPMI (with L-glutamine))
  2. gentleMACS Octo Dissociator with Heaters
  3. gentleMACS C Tubes
  4. Miltenyi Pre-Separation Filters (30um)
  5. DAPI (1mg/mL)
  6. Fixation Buffer (4% Paraformaldehyde + 1x PBS + 0.1% Tween 20)
  7. 1x PBS
  8. Permeabilization Buffer (Freshly prepared 70% Ethanol)
FOR FFPE NUCLEI PREP
  1. FFPE Block
  2. 100% Ethanol
  3. CitriSolv
  4. 1x PBS
  5. Miltenyi Pre-Separation Filters (30um)
  6. DAPI (1mg/mL)
  7. Fixation Buffer (4% Paraformaldehyde + 1x PBS + 0.1% Tween 20)
  8. PBS-T (1x PBS + 0.1% Tween 20)
  9. Permeabilization Buffer (Freshly prepared 70% Ethanol)
FOR CELL PREP
  1. Staining Buffer (1x PBS + 1% BSA)
  2. PBS-T (1x PBS + 0.1% Tween 20)
  3. Fixation Buffer (4% Paraformaldehyde + 1x PBS + 0.1% Tween 20)
  4. Permeabilization Buffer (Freshly prepared 70% Ethanol)
GENERAL REAGENTS & INSTRUMENTS
  1. Storage Buffer (Water + 1x Quenching Buffer + 0.1 Enhancer)
  2. Protector RNAse Inhibitor (Sigma-Aldrich Lot No: 3335399001)
Safety warnings
Attention
Use formaldehyde with caution as it 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)
Screenshot 2024-03-18 at 9.24.28 PM.png


Nuclei Preparation from Fresh Frozen Tissue
Nuclei Preparation from Fresh Frozen Tissue
FIXATION AND PERMEABILIZATION
Weight tissue to determine the volume of fixation buffer needed.
  • 2 mL fixation buffer per 25 mg tissue is required.
Place tissue on a pre-chilled glass petri dish maintained TemperatureOn ice and using a blade, mince tissue finely (enable passing through a 1mL wide-bore pipette tip)

Using a wide bore 1mL pipette, add the required fixation buffer depending on the amount of tissue.
Incubate at room temperature for 2-3 hours with intermittent shaking.Temperature25 °C

Centrifuge at 850xg for 5 minutes.
Gently remove supernatant without disturbing the pellet.
Add 2mL PBS per 25mg tissue to wash tissue.
Centrifuge at 850xg for 5 minutes.

Add 2mL permeabilization buffer per 25mg tissue and incubate overnight at Temperature4 °C to permeabilize.

Overnight
Centrifuge at 850xg for 5 minutes.
Remove supernatant without disturbing the pellet.
Add 2mL PBS per 25mg tissue to wash tissue.
Centrifuge at 850xg for 5 minutes.
TISSUE DISSOCIATION
Make the dissociation solution as follows:
  • Reconstitute 5mg Liberase (5401020001) with 1mL sterile water. Mix until fully dissolved and make single use aliquots for future use in Temperature-20 °C .

AB
ContentsFor ≤100mg tissue
RPMI (w L-glutamine)1840uL
Liberase160uL
Keep dissociation solution TemperatureOn ice until ready to proceed.
Warm the solution for 10 minutes at Temperature37 °C .

Add 2mL per ≤100mg pre-warmed dissociation solution to the sample.
Re-suspend and transfer 2mL of sample into each C tube.
Run the following program on the gentleMACS Octo dissociator with heaters ON:
AB
StepProgram
1Temp ON
2Spin 50 rpm, 40’ 0 “
3Spin 1000rpm, 30”
4Spin -1000rpm, 30”
5end

Ensure all tissue pieces are in suspension when flipping C tubes over for dissociation.
Once the program is done, ensure all tissue is dissociated before proceeding.
  • If there are large chunks of tissue that are undissociated, run a 50 rpm spin only for longer until chunks are dissociated.
Perform a quick pulse centrifuge (300xg) to collect all cells into the bottom of the tube.
Re-suspend and pass the dissociated tissue through a 30um filter to remove debris and tissue chunks.
Perform an additional wash by adding 2mL of PBS into tubes and passing through the same filter.
Count nuclei on countess or hemocytometer and record nuclei count.
  • Expect > 4 million nuclei per 25mg of tissue.
Stain with 1uL of DAPI (1mg/mL) per 5 million cells and sort DAPI+ nuclei.
Proceed immediately with the PERFF-seq: HCR Flow-FISH and Polymer Disassembly Protocol.

Stopping point:
Alternatively, samples can be resuspended in storage buffer (1mL per 5 million, see Materials section or 10XG protocol for more details).
Nuclei can be stored for up to 1 week at Temperature4 °C .
Pause
Nuclei Preparation from Formalin Fixed Paraffin Embedded (FFPE) Tissue (using S2 Genomics Singulator)
Nuclei Preparation from Formalin Fixed Paraffin Embedded (FFPE) Tissue (using S2 Genomics Singulator)

ISOLATION

Obtain 2 to 6 FFPE curls (Ideally 50uM to 70uM of thickness).
Total number and thickness of each curls will change based on how much tissue is embedded and desired number of nuclei.
Curls can be stored in a 1.5mL tube at Temperature4 °C for long term storage.

Pause
For each sample, pre-cool 2 NIC+ cartridges (S2 Genomics) be placing them in the fridge.
Prepare the following reagents in 15mL tubes (per sample).
  • 5mL Ethanol (100%)
  • 5mL CitriSolv (solvent)
  • 5mL of PBS (-Ca/Mg)
  • Empty 50mL tube for waste
Place reagent lines from S200+ Singulator Prototype into appropriate 15mL conicals.
a. Place Waste line into empty 50mL tube.
b. Place Solvent line into CitriSolv.
c. Place Ethanol line into Ethanol.
d. Place Buffer line into PBS.
Add curls to NIC+ cartridge.
Make sure the curls are on the bottom of the cartridge chamber.
Turn on Singulator and tablet.
Insert cartridge and start the FFPE extraction protocol.
The protocol (~50 minutes) will run deparaffination with CitriSolv, rehydration with Ethanol, and washes with PBS.
a. 15 minute CitriSolv incubation.
b. 7.5 minute CitriSolv incubation.
c. 7.5 minute CitriSolv incubation.
d. 1 minute 100% Ethanol incubation.
e. 1 minute 100% Ethanol incubation.
f. 1 minute 70% Ethanol incubation.
g. 1 minute 50% Ethanol incubation.
h. 1 minute 30% Ethanol incubation.
i. 3x PBS washes.
During the run, pre-cool Singulator 200 for subsequent nuclei extraction from tissue.
Upon completion of the protocol, remove the cartridge from the machine.
Using a P1000 pipette, remove the sample (will appear as small tissue pieces) and transfer into a 5mL conical tube TemperatureOn ice (Pipette gently, trying to not break further pieces of tissue during transfer).
Centrifuge at 1000g for 3 minutes at Temperature4 °C .
Remove the supernatant and resuspend in Amount500 µL of Nuclei Isolation Reagent (NIR).
Add Amount12.5 µL of Protector RNase Inhibitor (1U/uL final).
Then transfer Amount400 µL of NIR+ tissue to a new pre-cooled NIC+ cartridge.
Select FFPE Nuclei Isolation protocol.
Place cartridge in Singulator 200 and start protocol.
While Singulator is running (about 8 minutes), add 25uL of Protector RNase Inhibitor to 1mL of Nuclei Storage Reagent (NSR) (1U/uL final).
Upon completion of the run, remove the cartridge and pierce the foil to collect the isolated nuclei.
Transfer nuclei suspension into 5mL tubes TemperatureOn ice .
Centrifuge at 500xg for 5 minutes at Temperature4 °C .
Remove gently supernatant and resuspend in 1mL of NSR + Protector RNase Inhibitor (1U/uL final).
Filter nuclei suspension with Miltenyi Biotec Pre-Separation Filters (30um).
Count nuclei using DAPI.
Trypan can be used when counting with brightfield, but DAPI will yield more accurate counts.
FIXATION AND PERMEABILIZATION
Spin nuclei at 850xg for 5 minutes at Temperature4 °C and gently remove supernatant.
Resuspend and add fixation buffer to fix cells at 1M/mL concentration.
Incubate at room temperature for one hour or Temperature4 °C overnight.

Overnight
After incubating, centrifuge at 850xg for 5 minutes and aspirate supernatant.
Resuspend in 1x PBS + 0.5% BSA + 0.2U/uL Protector RNase Inhibitor (final) at 1M/1mL concentration TemperatureOn ice .

Centrifuge at 850xg for 5 minutes at Temperature4 °C and aspirate supernatant.
Resuspend in 1x PBS + 0.5% BSA + 0.2U/uL Protector RNase Inhibitor (final) at 1M/1mL concentration TemperatureOn ice .

Filter nuclei suspension through a 35um FACS tube cap filter (blue) or Miltenyi Biotec Pre-Separation Filters (30um).
Stain with 1uL of DAPI (1mg/mL) per 5 million cells and sort DAPI+ nuclei.
DAPI+ nuclei can be sorted (suggested nozzle of 100um) into 150uL of 1x PBS + 0.5% BSA + 0.2U/uL Protector RNase Inhibitor (final).
Use 1.5mL Lobind Eppendorf as collection tube.
Centrifuge sorted nuclei at 850xg for 5 minutes at Temperature4 °C and gently remove supernatant.
Add ice cold permeabilization buffer to nuclei at 1M/mL concentration.
Incubate at Temperature4 °C overnight.
Overnight
Remove samples from Temperature4 °C and centrifuge at 850xg for 5 minutes.
Add 1M/mL of PBST and count nuclei to ensure minimal cell loss post fixation and permeabilization.
Nuclei can be split into 1 million - 5 million nuclei aliquots for HCR Flow-FISH experiments.
Centrifuge at 850xg for 5 minutes.
Repeat wash with 500uL of PBST.
Proceed immediately with the PERFF-seq: HCR Flow-FISH and Polymer Disassembly Protocol.
Stopping point:
Alternatively, samples can be resuspended in storage buffer (1mL per 5 million, see Materials section or 10XG protocol for more details).
Nuclei can be stored for up to 1 week at Temperature4 °C .
Overnight
Preparation of Peripheral Blood Mononuclear Cells (PBMCs) & Cell Lines
Preparation of Peripheral Blood Mononuclear Cells (PBMCs) & Cell Lines
ANTIBODY STAINING (Optional): For Use in conjunction with HCR FlowFISH

Note
For enrichment applications based on HCR FlowFISH and surface antibody staining, we recommend performing surface staining before fixing and permeabilizing cells.
  • We note that surface antibodies conjugated to synthetic dyes result in the most robust signal when used in conjunction with our HCR FlowFISH protocol.
  • For downstream dead cell exclusion, a fixable viability dye can be used prior to fixation and permeabilization.

Optional
Re-suspend cells with 45uL of staining buffer. Add 5uL of Fc block to cells and incubate at Temperature4 °C for 15 minutes.

Make antibody cocktail based on manufacturer recommendations and re-suspend cells.
Incubate in dark at Temperature4 °C for 20 minutes.
Wash 2 times with staining buffer to remove unbound antibodies.
If using a fixable viability dye, staining can be done here.
FIXATION AND PERMEABILIZATION
Add fixation buffer to fix cells at a 1M/mL concentration.
  • For example, re-suspend 50M cells in 50mL of fixation solution
Incubate at room temperature for one hour or Temperature4 °C overnight.
After incubating, centrifuge at 850xg for 5 minutes.
Wash 2 times with PBS-T at 1M/mL concentration.
Add ice cold permeabilization buffer to cells at 1M/mL concentration.
Incubate at Temperature4 °C overnight.

Overnight
Remove samples from Temperature4 °C and centrifuge at 850xg for 5 minutes.

Add 1M/mL of PBST and count cells to ensure minimal cell loss post fixation and permeabilization.
Cells can be split into 1 million - 5 million cell aliquots for HCR Flow-FISH experiments.
Centrifuge at 850xg for 5 minutes.
Repeat wash with 500uL of PBST.
Proceed immediately with the PERFF-seq: HCR Flow-FISH and Polymer Disassembly 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 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. Tissue Fixation & Dissociation for Chromium Fixed RNA Profiling. 10x Genomics
4. PERFF-seq: HCR Flow-FISH and Polymer Disassembly Protocol