Jun 27, 2025

Public workspaceMultiplexed Fixed Single Cell RNA Sequencing of frozen human post mortem brain

DOI
dx.doi.org/10.17504/protocols.io.5qpvok6b9l4o/v1
  • Christoph Much1,
  • Joshua Laß1,
  • Mohsin Shafiq2,
  • Christine Klein1,
  • Markus Glatzel2,
  • Joanne Trinh1
  • 1Institute of Neurogenetics, University of Luebeck;
  • 2Institute of Neuropathology, University Medical Center Hamburg-Eppendorf
Christoph Much
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DOI: https://dx.doi.org/10.17504/protocols.io.5qpvok6b9l4o/v1
Protocol Citation: Christoph Much, Joshua Laß, Mohsin Shafiq, Christine Klein, Markus Glatzel, Joanne Trinh 2025. Multiplexed Fixed Single Cell RNA Sequencing of frozen human post mortem brain. protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvok6b9l4o/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: In development
We are still developing and optimizing this protocol
Created: September 18, 2024
Last Modified: June 27, 2025
Protocol Integer ID: 107816
Keywords: Short-read sequencing, 10X Genomics, Multiplexing, Single-cell sequencing, FACS Sorting, Nuclei, multiplexed fixed single cell rna sequencing, frozen human post mortem brain this protocol, frozen human post mortem brain, using 10x genomic, 10x genomics for the library preparation, rna, infectious tissue, tissue, 10x genomic
Disclaimer
In development
We are still developing and optimizing this protocol.
Abstract
This protocol describes the processing of post-mortem frozen brain tissue for fixed single-cell sequencing using 10x Genomics for the library preparation. This protocol can be applied and generalized for infectious tissues that need to be fixed before processing.
Materials
10x Genomics Kits:
Chromium Nuclei Isolation Kit with RNase Inhibitor, 16 rxns (Ref: 1000494)
Chromium Fixed RNA Kit, Human Transcriptome, 4rxns x 4 BC (Ref: 1000475)
Chromium Next GEM Chip Q Single Cell Kit, 16 rxns (Ref: 1000422)
Chromium Next GEM Single Cell Fixed RNA Sample Preparation Kit, 16 rxns (Ref: 1000414)
Dual Index Kit TS Set A, 96 rxn (Ref: 1000251)

Additional Reagents and Plastic Wear not included:
DNA LoBind Tubes 1,5ml (Eppendorf; Ref: 022431021)
PCR Tubes 0.2ml 8-tube strips (Eppendorf; Ref: 951010022)
15ml Centrifuge Tube (Sarstedt; Ref: 62.554.016)
Pre-Separation Filters (30µm) ( Milentyi Biotec; Ref: 130-041-407)
Nuclease-free Water
Low TE Buffer (10mM Tris-HCL pH 8.0 0.1mM EDTA) (Thermo Fisher; Ref: 12090-105)
Tween 20 Surfact-Amps Detergent Solution (10%) (Thermo Fisher; Ref: 28320)
SPRIselect Reagent Kit (Beckman Coulter; Ref: B23318)
Ethanol (Millipore Sigma; Ref: E7023-500ml)
Gylercol (Millipore Sigma; Ref: G5516)
Buffer EB (Qiagen; Ref: 19086)
37% Formaldehyde (Thermo Fisher; Ref: BP531-500)
Bovine Serum Albumin In DPBS (10%) (Millipore Sigma; Ref: A1595)
DAPI 10mg (Thermo Fisher; Ref: D3571)
High Sensitivity D5000: ScreenTape (Agilent; Ref: 5067-5592)
High Sensitivity D500: Reagents (Agilent; Ref: 5067-5593)

Equipment:
10x Magnetic Seperator
10x Chromium iX/X
FACS Sorter
Brightfield-Microscope
Agilent TapeStation
Mini Centrifuge
Thermocycler (100µl Volume Capacity)
Eppendorf 1.5ml ThermoMixer C
One of the Following Illumina Sequencers: iSeq. MiSeq, NextSeq 500/550, NextSeq 1000/2000, NovaSeq
Troubleshooting
Before start
For this protocol, frozen brain tissue from liquid nitrogen is recommended. The samples may be highly variable in cell counts and quality due to various dependencies, such as disease status, the time it took to transfer the samples into liquid nitrogen after dissection, the time elapsed between the patient's death and the dissection, and the brain region being observed. This protocol was performed using striatal tissue.
Using ~30mg of tissue led to 100.000-400.000 cells counted in FACS, which resulted in 2000-13,000 unique cells sequenced.
To get an estimate on the quality of the sample, a percentage was sorted to single cell fraction with FACS.
5-20% or greater single cells is optimal, and it is safe to continue. A higher yield of 6,000-13,000 cells in the end may be expected.
2-5% Single Cells are of average quality to continue, if the result of 3,000-6,000 Cells are enough for analysis.
Less than 2% indicates a poor cell quality, and a low yield of reads with poor quality is expected even if a high number of Cells (e.g. 200.000) have been collected in the Single Cell Fraction. Continue at own risk.

Nuclei Isolation
2h 15m 40s
Obtain the 10x Nuclei Isolation Kit, Nuclease-Free Water, PBS, and 10x BSA.
Pre-chill the centrifuge to Temperature4 °C .

Temperature
Obtain materials and reagents as listed:



10m
Prepare Lysis Buffer and place TemperatureOn ice .






5m
Prepare Debris Removal Buffer and place TemperatureOn ice .




5m
Prepare the Wash and Resuspension Buffer and place TemperatureOn ice .




5m
Nuclei Isolation
1h
Cut Amount30 mg of the frozen tissue on dry ice and put it in the sample dissociation tube.

10m
Add 200µl of Lysis Buffer to the tissueTemperatureOn ice .

1m
Homogenize the tissue until no clumps remain, then add an additional Amount300 µL of Lysis Buffer and mix 10 times with a pipette.

2m
Incubate forDuration00:10:00 TemperatureOn ice .

10m
Transfer the lysate to the Nuclei Isolation Column.
1m
Centrifuge Centrifigation16.000 rcf for Duration00:00:20 .

20s
Keep the Collection Tube and discard the column.
Resuspend the nuclei by vortexing the Collection Tube forDuration00:00:10 atCentrifigation3.200 rpm .

1m
Centrifuge the Collection Tube Duration00:03:00 at Centrifigation500 rcf .
3m
Discard the supernatant.
Strongly recommended: a small amount of Amount200 µL may be left behind if a low yield is expected.

2m
Add Amount500 µL Debris Removal Reagent and resuspend by pipetting.

1m
Centrifuge at Centrifigation700 rcf forDuration00:10:00 .

10m
Discard the supernatant.

Note: The nuclei will be sitting at the bottom-side of the tube, and there will be a lot of debris/fat on the upper side of the tube. Gently take off the supernatant from the side of the wall, without going too low to take up the nuclei.
Strongly recommended: an amount of Amount200 µL may be left behind if a low yield is expected.
2m
Resuspend in Amount1 mL Wash and Resuspension Buffer.
1m
Centrifuge atCentrifigation500 rcf for Duration00:05:00 .

5m
Discard the supernatant.
Strongly recommended: a small amount of Amount100 µL may be left behind if a low yield is expected.
1m
Add Amount1 mL of Fixation Buffer, resuspend by pipetting and leave at Temperature4 °C for Duration16:00:00 -Duration24:00:00 .




20s
FACS sorting
2h 27m
Cell sorting is mandatory, as ~95% of the pellet will be debris or clumps

If the cells have been fixed overnight, continue with the cell sorting.
Note: Ensure that all samples have been fixed for the same duration.
Prepare the buffers and reagents and place TemperatureOn ice :
-Quenching Buffer:

-PBS+2% BSA:
Amount4 mL PBS
Amount1 mL 10% BSA

-300µM DAPI:
Dilute Amount300 µL 1mM DAPI stock solution with Amount700 µL of PBS.

5m
FACS Sorting
Centrifuge fixed cells atCentrifigation900 rcf Duration00:05:00 at TemperatureRoom temperature .

5m
Take off the supernatant.

Note: the pellet should be more compact now, and the full removal of supernatant is possible.
2m
Resuspend the pellet in Amount500 µL cold Quenching Buffer and incubate Duration00:05:00 TemperatureOn ice .

5m
Centrifuge cells atCentrifigation900 rcf Duration00:05:00 at TemperatureRoom temperature .
5m
Remove supernatant and resuspend in Amount200 µL -Amount500 µL PBS+2% BSA.

Note: if the cell suspension is too dense for FACS sorting, adjust with PBS+2%BSA to achieve a sort rate of ~10000 events/second during sorting to avoid clotting of the machine.

2m
Pass the sample through a 30µm strainer directly into the FACS sorting tubes.
2m
Add Amount5 µL 300µM DAPI to Amount1000 µL of cell suspension.

1m
Sort the single DAPI positive events to a fresh 1.5 ml LoBind Tube and place TemperatureOn ice .

Note: Each sample will take about 30 minutes to sort
Depending on tissue quality, expect 50000-300000 single nuclei
If it is less than 100000 nuclei, it is not recommended to continue as there is more nuclei loss expected during the preparation of the samples

2h

Example of a good sort with a high fraction of 13.6% single DAPI positive events.

Probe Hybridization
16h 35m
Note: Library preparation is following the standard 10x Genomics "Chromium Fixed RNA Profiling Reagent Kits for Multiplexed Samples" protocol with some tweaks for maximizing nuclei yield:
https://www.10xgenomics.com/support/flex-gene-expression/documentation/steps/library-prep/chromium-single-cell-gene-expression-flex-reagent-kits-for-multiplexed-samples


Prepare the following reagents as listed:


5m
Prepare the Hyb Mix at TemperatureRoom temperature :



2m
Incubate Hyb Mix at Temperature42 °C for Duration00:05:00 .

5m
Centrifuge fixed nuclei at Centrifigation900 x g for Duration00:05:00 and take off the Supernatant.

Very Important: When removing the supernatant, leave about Amount20 µL behind. This drastically increases the yield and does not affect the hybridization performance.

5m
Resuspend the pellet in Amount80 µL Hyb Mix and transfer to a 8-strip tube. Keep the samples at TemperatureRoom temperature

2m
Add Amount20 µL of Unique Human WTA-Probe Barcode per sample and gently mix by pipetting.

1m
Incubate samples for 16-24h at Temperature42 °C in a thermocycler with heated lid.
Continue with counting and GEM generation the next day.

16h
Count the nuclei using Amount5 µL .
Note: A high nuclei loss can occur here. For example, starting with 150,000-400,000 nuclei after FACS can result in 35,000-140,000 after probe hybridization and then a subsequent final read count of 3,500-13,000 per sample.

15m
GEM Generation and Barcoding
3h 34m 30s
Prepare the following reagents and materials as listed:




5m
Prepare the following buffer:

Use the upper table, if multiplexing 4 samples, and the lower table for up
to 16 samples


5m
Pooling and Washing of the samples
Pool the samples, using the cell count you got after hybrdization in a 15ml Falcon.

NOTE: Usually, take all nuclei of the sample with the lowest concentration, and an equal amount of nuclei from the other samples to create one pool, where there are similar amount of cells from each sample.
If one sample has way lower concentration than the others, it also worked out to Pool the full amount of all samples. But expect less reads from the sample with the low concentration.
5m
Add Amount3 mL Post-Hyb Wash Buffer and mix by inverting 5 times.

1m
Centrifuge the pool at Centrifigation900 x g for Duration00:05:00 and take off the supernatant.
5m
Resuspend cell pellet in Amount1 mL Post-Hyb Wash Buffer and transfer to a 1.5ml Tube.

1m
Incubate at Temperature42 °C for Duration00:10:00 .

10m
Centrifuge at Centrifigation900 x g for Duration00:05:00 and take off the supernatant.
5m
Resuspend cell pellet in Amount0.5 mL Post-Hyb Wash Buffer.
1m
Incubate at Temperature42 °C for Duration00:10:00 .
10m
Critical step: Filter the nuclei through a 30µm Strainer into a new 1.5ml Tube, to avoid clumps going into the GEM generation.
1m
Centrifuge at Centrifigation900 x g for Duration00:05:00 and take off the supernatant.
5m
Prepare Post-Hyb Resuspension Buffer and maintain at Temperature4 °C :



2m
Resuspend the pellet in an appropriate amount of Post-Hyb Resuspension Buffer and place TemperatureOn ice :



2m
Count the nuclei and calculate the total amount.

Note: The lowest concentration should be 2000 nuclei/µl so 1.000.000 in total. If there are less nuclei, centrifuge at Centrifigation900 x g for Duration00:05:00 and remove as much supernatant as needed, to achieve a concentration of 2000 nuclei/µl.
For example if you have 100.000 Nuclei in 500µl, remove 450µl of supernatant after centrifugation to achieve a concentration of 2000 Nuclei/µl in 50µl.
5m
Prepare GEM Master Mix + Sample Dilution
Prepare Master Mix TemperatureOn ice . Pipette mix 15x and centrifuge briefly.



2m
In a PCR 8-tube strip, prepare a dilution depending on the cell stock concentration and target cell recovery.

Note: If you have a concentration of 2000 Cells/µl and aim for a Cell Recovery of 40000 cells, dilute Amount33 µL of Cell Stock with Amount7 µL Post-Hyb Resupension Buffer.

2m
Add Amount35 µL of prepared GEM Master Mix into each tube containing the cell dilution and immediately proceed to the next step.

Note: Don´t mix the sample at this point.
1m
Assembly and Loading of the Next GEM Chip Q


  • Attach the gasket, with the smooth side facing the chip holder
  • Open the chip holder
  • Remove the chip from the bag, align the chip with the notch on the left side of the Holder and secure the chip with the clip on the right side
  • The chip is now ready for loading
  • Do not touch the smooth side of the gasket
2m
When loading the chip, ensure the chip lays flat and raise the pipette tip as you dispense. This should take Duration00:00:05 .



Add 50% glycerol solution to each unused well:


  • 70 μl in each unused well in row labeled 1
  • 50 μl in each unused well in row labeled 2
  • 150 μl in each unused well in row labeled 3


2m



Prepare Gel Beads:
  • Snap the tube strip holder with the Gel Bead strip into a 10x Vortex Adapter. Vortex Duration00:00:30 .
  • Centrifuge the Gel Bead strip for Duration00:00:05 . Confirm there are no bubbles at the bottom of the tubes & the liquid levels are even.
  • Place the Gel Bead strip back in the holder. Secure the holder lid.


5m


Load Row 1:
  • With pipette set to 70 μl, gently pipette mix the GEM Master Mix + Sample 15x.
  • Using the same pipette tips, dispense Amount70 µL GEM Master Mix + Sample into the bottom center of wells in row labeled 1 without introducing bubbles.

2m


Load Row Labeled 2:
  • Puncture the foil seal of the Gel Bead tubes. Slowly aspirate Amount50 µL Gel Beads.
  • Dispense into the wells in row labeled 2 without introducing bubbles.
  • Wait Duration00:01:00 .


2m


Load Row Labeled 3:

  • Dispense Amount45 µL Partitioning Oil into the wells in row labeled 3 from a reagent reservoir.

2m


Prepare for Run:
  • Close the lid (gasket already attached). DO NOT touch the smooth side of the gasket. DO NOT press down on the top of the gasket.
1m
Run the Chromium X/iX
  • Press the eject button on the Chromium X to eject the tray. If the eject button is not touched within 1 min, tray will close automatically. System requires a few seconds before the tray can be ejected again.
  • Place the assembled chip with the gasket in the tray, ensuring that the chip stays horizontal. Press the button to retract the tray.
  • Press the play button.
  • At completion of the run (Duration00:05:30 ), Chromium X/iX will chime. Immediately proceed to the next step.

5m 30s
Transfer GEMs
  • Place a tube strip on ice.
  • Press the eject button of the Chromium X/iX and remove the chip.
  • Discard the gasket. Open the chip holder. Fold the lid back until it clicks to expose the wells at 45 degrees.
  • Check the volume in rows labeled 1-2. Abnormally high volume in any well indicates a clog.
  • Slowly aspirate Amount100 µL GEMs from the lowest points of the recovery wells
in the top row labeled 3, without creating a seal between the tips and the bottom of the wells.
  • Withdraw pipette tips from the wells. GEMs should appear opaque and uniform across all channels.
  • Over the course of Duration00:00:20 , dispense GEMs into the tube strip on ice with the pipette tips against the sidewalls of the tubes.

5m
GEM Incubation
Incubate in a Thermal Cycler using the following protocol:


Note: Store at Temperature4 °C for up to a week, or proceed immediately to the next step.

2h
GEM Recovery and Pre-Amplification
1h 18m 30s
Prepare the following reagents and materials as listed:



5m
Post-Gem Incubation-Recovery
Add Amount125 µL Recovery Agent to each sample at TemperatureRoom temperature . DO NOT pipette mix or vortex the biphasic mixture.

2m
Mix by inverting 5x and incubate for Duration00:02:00 .

2m
Centrifuge briefly and slowly remove Amount125 µL Recovery Agent (pink phase).

Note: A small amount of Amount20 µL Recovery Agent is normal and does not affect the performance.

2m
Pre-Amplification PCR
Prepare Pre-Amplification Mix on ice. Vortex and centrifuge briefly.



2m
Add Amount35 µL Pre-Amplification Mix to the aqueous sample.

1m
Mix by inverting 8x and centrifuge briefly.
1m
Incubate in a Thermocycler using the following protocol:




40m
Store at Temperature4 °C for up to Duration72:00:00 or Temperature-20 °C for 1 week, or proceed to the next step.

DNA Cleanup- SPRISelect
Prepare the Elution Solution. Vortex briefly.




2m
Centrifuge the sample for Duration00:00:30 in a microcentrifuge and transferAmount70 µL of the upper layer to a fresh 8-tube strip.

Note: If a low yield is expected, transfer all of the upper layer and adjust the Volume of SPRIselect accordingly.

2m
Vortex to resuspend the SPRIselect reagent. Add Amount126 µL SPRIselect reagent (1.8X) to each sample and pipette mix 15x.
Incubate for Duration00:05:00 atTemperatureRoom temperature .

5m
Place on the magnet "high" until the solution clears.
2m
Remove the supernatant and wash the beads with Amount200 µL 80% Ethanol.
Wait for Duration00:00:30 .

30s
Remove the ethanol and repeat the previous step for a total of 2 washes.
5m
Centrifuge briefly and place on the magnet "low".
Remove any remaining ethanol, but do not let the beads dry out.
1m
Remove from the magnet. Add Amount101 µL Elution Solution. WaitDuration00:01:00 before resuspending. Pipette mix 15x.

2m
Incubate Duration00:02:00 at TemperatureRoom temperature .

2m
Place the tube strip on the magnet "High" until the solution clears.
1m
Transfer Amount100 µL supernatant to a new 8-tube strip.

1m
Store at Temperature4 °C for up to Duration72:00:00 or Temperature-20 °C for 1 week, or proceed to the next step.
Library Construction
1h 30m 30s
Prepare the following reagents and materials as listed:



5m
Choose an appropriate index so there are no overlaps during one sequencing run.
Prepare the Sample Index PCR Mix TemperatureOn ice :



5m
Transfer only Amount20 µL of the cleaned up sample to a new strip and add Amount60 µL of the prepared Sample Index PCR Mix.

2m
Add Amount20 µL of an individual Dual Index TS Set A to each sample. Pipette mix 5x.

1m
Incubate in a Thermal Cycler using the following protocol:


Depending on the targeted cell recovery, choose the number of cycles from the Table below.
Note: For analyzing 10.000 cells/barcode (40.000 in total), choose 10 cycles for the PCR program.


30m
Store atTemperature4 °C for ≤Duration72:00:00 , or proceed to the next step.

Post Sample Index PCR Size Selection – SPRIselect
Vortex to resuspend the SPRIselect reagent. Add Amount100 µL SPRIselect Reagent (1.0X) to each sample. Pipette mix 15x and incubate for Duration00:05:00 at TemperatureRoom temperature .

5m
Place on the magnet "high" until the solution clears.
2m
Remove the supernatant and wash the beads with Amount200 µL 80% ethanol.
Wait for Duration00:00:30 .
30s
Remove the ethanol and repeat the previous step for a total of 2 washes.
5m
Centrifuge briefly and place on the magnet "low".
Remove any remaining Ethanol, but do not let the beads dry out.
2m
Remove from the magnet. Add Amount41 µL Buffer EB. Pipette mix 15x.

1m
Place the tube strip on the magnet "low" until the solution clears.
1m
Transfer Amount40 µL to a new tube strip.
The Library is now finished and can be prepared for Illumina Sequencing as needed.
1m
Store at Temperature4 °C for up to Duration72:00:00 or at Temperature-20 °C for long-term storage.

Post Library Construction QC
Run Amount1 µL sample at 1:80 dilution on an Agilent Bioanalyzer High Sensitivity chip or on a Tapestation D5000 Screen Tape Assays.
Select the region between 150-300 bp to determine average size of the library.

30m