Jan 16, 2024

Public workspacesnPATHO-seq

DOI
dx.doi.org/10.17504/protocols.io.8epv5x58dg1b/v1
  • 1Garvan Medical Institute;
  • 2Adelaide Centre for Epigenetics, University of Adelaide;
  • 3University of Adelaide
  • Jasmine Plummer: Center for Spatial OMICs, St Jude Children’s Research Hospital;
Michael Roach
Open access
DOI: dx.doi.org/10.17504/protocols.io.8epv5x58dg1b/v1
Protocol Citation: Tony Wang, Michael Roach, Jasmine Plummer, Luciano G Martelotto 2024. snPATHO-seq. protocols.io https://dx.doi.org/10.17504/protocols.io.8epv5x58dg1b/v1
Manuscript citation:
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: January 10, 2024
Last Modified: January 16, 2024
Protocol Integer ID: 93229
Abstract
Formalin-fixed paraffin-embedded (FFPE) samples are valuable but under-utilised in single-cell omics research due to their low DNA and RNA quality. Leveraging recent single-cell genomic technology advances, we introduce snPATHO-seq: a versatile method to derive high-quality single-nucleus transcriptomic data from FFPE samples.
Materials
Reagents and consumables
  • Ethanol
  • Xylene
  • Nuclease Free water
  • 1x Phosphate Buffer Saline (PBS, Ca2+ and Mg2+ free)
  • Liberase TM or TH (Roche)
  • Collagenase D or P (Roche)
  • Hyaluronidase (CAS 37326-33-3, Calbiochem)
  • RPMI1640 (Gibco)
  • EZ Lysis Buffer (Sigma)
  • 10% BSA (MACS‱ BSA Stock Solution, Miltenyi)
  • Glycerol 50%
  • RNAse Inhibitor (RiboLock from Thermo or RNA Protector from Roche)
  • (Optional) 4',6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI, ThermoFisher)
  • 40 um and 70 um pluriStrainer filters (pluriSelect) (MACS SmartStrainers are also possible)
  • 25 G needle

Equipment
  • Thermomixer with adjustable shaking (Eppendorf)
  • Swinging bucket refrigerated centrifuge
Paraffin removal
Paraffin removal
Cut 1 or 2 approximately 25 μm-thick sections (punches are also possible) and transfer to a 1.5 mL Eppendorf tube.

Note
Store dry at 4°C if not used immediately. To keep it dry store in a container with silica beads. You can use the cylinder with silica beads that comes with 10x Genomics chips.

Add Amount1 mL Xylene and incubate for Duration00:10:00 at TemperatureRoom temperature or Temperature50-55 °C

Note
Optional: heat at 50-55°C for 10 mins for the first xylene wash. This has proven to be very helpful in removing the paraffin more efficiently, in particular with fatty tissue.

10m
Carefully remove Xylene without disturbing the sample.
Repeat steps 2 and 3 two more times:
Add Amount1 mL Xylene , incubate Duration00:10:00 at TemperatureRoom temperature , remove xylene
Add Amount1 mL Xylene , incubate Duration00:10:00 at TemperatureRoom temperature , remove xylene

20m
Rehydration
Rehydration

Note
OVERVIEW: Wash sections with sequential ethanol immersions: 2x 100%, 1x 70%, 1x 50%, 1x 30%). Lastly, wash with RPMI1640.

Add Amount1 mL 100% ethanol and incubate for Duration00:01:00 at TemperatureRoom temperature , then carefully remove ethanol without disturbing the sections/punches.

1m
Repeat step 5: add Amount1 mL 100% ethanol , incubate for Duration00:01:00 at TemperatureRoom temperature , remove ethanol wash
1m
Add Amount1 mL 70% ethanol , incubate for Duration00:01:00 at TemperatureRoom temperature , remove ethanol wash
1m
Add Amount1 mL 50% ethanol , incubate for Duration00:01:00 at TemperatureRoom temperature , remove ethanol wash
1m
Add Amount1 mL 30% ethanol , incubate for Duration00:01:00 at TemperatureRoom temperature , remove ethanol wash
1m
Add Amount1 mL RPMI1640 , incubate for Duration00:01:00 at TemperatureRoom temperature , carefully remove RPMI1640 wash
1m
Tissue dissociation
Tissue dissociation
Prepare Amount1 mL Dissociation Solution :
  • Amount1 mL RPMI1640
  • Concentration0.25-1 mg/mL Liberase TM(*)
  • Concentration0.25-1 mg/mL 10x Collagenase D(**)
  • Concentration0.25-1 mg/mL Hyaluronidase
  • Concentration1 U/uL RNAse Inhibitor

Add this Amount1 mL Dissociation Solution to the sections/punches.

Note
(*) Liberase TH is very good enzyme too and can be used alone at 1-2.5 mg/mL.
(**) Collagenase P (Roche) works fine as well.

IMPORTANT: I strongly recommend testing what concentration of enzymes works best for your tissue of interest. In our experience, Liberase TH alone at 1 mg/mL or the trio at 1 mg/mL Liberase TM + 1 mg/mL Collagenase D + 0.5 mg/mL Hyaluronidase works well with most of the samples we tested.

  • Pro tip: Add 200 μL of the enzymatic cocktail and mince using a pestle for at least 10 strokes, then complete to 1 mL with the rest of the cocktail mix.

  • Optional Pro tip: Before digestion, add 100 μL of digestion mix and homogenize the sample using a douncer/pestle by stroking 10-20 times. This helps in the digestion step. Then top up with the rest of the digestion mix.

Digest tissue for 45-60 mins(*) at 37°C in a Thermomixer at 800 RPM.
Shaker800 rpm, 37°C, 00:45:00 in Thermomixer

Note
(*) Some blocks require longer digestion time. Inspect visually and help dissociation by pipetting up and down with a P1000 pipette.

IMPORTANT: Dissociation does not need to be complete; the objective here is to loosen up the material to facilitate the nuclei release. Dissociation completeness varies from block to block. Tissue does not need to be fully digested.


45m
Lysing the cells
Lysing the cells

Note
OVERVIEW: Wash with lysis buffer. Resuspend and homogenize in small volume of lysis buffer. Add rest of lysis buffer and homogenise several times.

Add Amount400 µL Ez Lysis Buffer to the sample and mix by inverting 5 times, then centrifuge Centrifigation850 rcf, 4°C, 00:05:00
5m
Prepare Amount2 mL Lysis Solution as follows:
  • Amount2 mL Ez Lysis buffer
  • Concentration2 % (v/v) BSA
  • Concentration1 U/uL RNAse Inhibitor

Remove supernatant and add Amount250 µL Lysis Solution (from step 14)

Homogenize the sample using a douncer/pestle by stroking 10-20 times (or as needed).
Add a further Amount750 µL Lysis Solution (from step 14)
Homogenize by pipetting using a P1000 pipette (10 times), then incubate TemperatureOn ice for Duration00:05:00

5m
Repeat step 18: pipette 10 times and incubate TemperatureOn ice for Duration00:05:00
5m
Optional but very useful when possible: If the dissociation and disaggregation look
almost complete (i.e. only very small chunks of undigested tissue or fat are visible to the naked eye) gently pass the sample through a 25G needle for 20 times (avoid foaming). It is essential to ensure that no large chunks remain before passing though needle. If large chunks or fat remain the needle will definitely block, so just skip this step. This optional step will increase the nuclei release.

Cleaning the nuclei
Cleaning the nuclei

Note
OVERVIEW: Filter (large pore size). Wash with lysis solution. Wash with PBS. Filter (small pore size).

Prepare Amount5 mL Wash Solution as follows:
  • Amount5 mL 0.5x PBS
  • Concentration0.02 % volume BSA
Pass the sample through a 70 μm PluriStrainer filter to remove large chunks of undigested tissue.

Note
Do not use a FLOWMI cell strainer!

Centrifuge the flow-through Centrifigation850 rcf, 4°C, 00:05:00
5m
Remove supernatant and resuspend with Amount800 µL Lysis solution (from step 14)
Centrifuge Centrifigation850 rcf, 4°C, 00:05:00
5m
Remove supernatant and resuspend in Amount500-1000 µL Wash solution (from step 21)

Note
Resuspension volume can vary depending on the pellet size.

Centrifuge Centrifigation850 rcf, 4°C, 00:05:00
5m
Repeat wash steps 26 and 27: remove supernatant, resuspend in Amount500-1000 µL Wash solution (from step 21) and centrifuge Centrifigation850 rcf, 4°C, 00:05:00
5m
  • Remove supernatant and resuspend in Amount500-1000 µL Wash solution (from step 21)
  • Pass sample through a 40 μm PluriStrainer filter

Note
Do not use a FLOWMI cell strainer!

Using the nuclei
Using the nuclei
15m
Count using Luna-FX7 or similar based on dual-fluorescence such as AO/PI.

Note
Cycling conditions for Index PCR might need to be optimized per sample to obtain a final library that falls within ~50-200 nM. For nuclei derived from FFPE blocks, we typically use 1-2 additional cycles during indexing to start with. If the library does not reach the recommended range but the Bionalyzer/Fragment Analyzer/Tapestation traces look as expected (single peak at ~265 bp), then do not add additional cycles. If you see signs of under/over amplification in the traces, then adjust cycling accordingly.

Step case

Steps for immediate usage
1 step

Rest on wet ice for immediate FACS cytometry analysis/sorting, or proceed to the Chromium X run using Chromium Fix RNA Profiling (10x Genomics) following the user guide for singleplexed or multiplexed samples accordingly.