Sep 29, 2023

Public workspacePurification of human cortex excitatory neuron nuclei from fetal and postnatal tissue using fluorescent activated nuclei sorting (FANS) in combination with a SATB2 antibody.

 Forked from Fluorescence-activated nuclei sorting (FANS) on human post-mortem cortex tissue enabling the isolation of distinct neural cell populations for multiple omic profiling
DOI
dx.doi.org/10.17504/protocols.io.n92ldz9d8v5b/v1
  • Jonathan P Davies1,
  • Stefania S S Policicchio1,
  • Barry Chioza1,
  • Gina Commin1,
  • Joe Burrage1,
  • Emma L Dempster1,
  • Jonathan Mill1
  • 1University of Exeter Medical School, Exeter, UK
  • Complex Disease Epigenetics Group
Jonathan P Davies
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DOI: https://dx.doi.org/10.17504/protocols.io.n92ldz9d8v5b/v1
Protocol Citation: Jonathan P Davies, Stefania S S Policicchio, Barry Chioza, Gina Commin, Joe Burrage, Emma L Dempster, Jonathan Mill 2023. Purification of human cortex excitatory neuron nuclei from fetal and postnatal tissue using fluorescent activated nuclei sorting (FANS) in combination with a SATB2 antibody.. protocols.io https://dx.doi.org/10.17504/protocols.io.n92ldz9d8v5b/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: January 27, 2022
Last Modified: September 29, 2023
Protocol Integer ID: 57511
Keywords: FANS, post-mortem brain, nuclei, flow cytometry, anti-NeuN, nuclei sorting, anti-SATB2, fetal, neurodevelopment, FACS, neuron, development, nuclei from multiple different human brain cell, optimal marker for neuronal nuclei, purification of human cortex excitatory neuron nuclei, neuronal nuclei, nuclei from excitatory neuron, cellular composition in regulatory genomic study, multiple different human brain cell, human cortex excitatory neuron nuclei, heterogeneous mix of different neural cell type, epigenetic process, satb2 antibody, fetal cortex, different neural cell type, postnatal cortex, regulatory genomic study, open chromatin analysis, gene regulation, current analyses of the human brain, genome, human brain, postnatal tissue, rna, activated nuclei, specific patterns of gene regulation, transcriptional variation in health, specific cell, purified populations of nuclei, transcriptional variation, gene expression, cellular composition, determining cell type, dna, excitatory neuron, neuron, nuclei, cortex, cell type
Funders Acknowledgements:
Alzheimer’s Research UK (ARUK)
Grant ID: ARUK-PPG2018A-010
Simons Foundation for Autism Research (SFARI)
Grant ID: 573312
Medical Research Council UK (MRC UK)
Grant ID: MR/R005176/1
Abstract
Increased understanding of the functional complexity of the genome has led to growing recognition about the role of epigenetic/transcriptional variation in health and disease. Current analyses of the human brain, however, are limited by the use of “bulk” tissue, comprising a heterogeneous mix of different neural cell types. Because epigenetic processes play a critical role in determining cell type-specific patterns of gene regulation it is important to consider cellular composition in regulatory genomic studies of human post-mortem tissue, and there is a need for methods to purify populations of specific cell-types. This protocol builds on a previous protocol that uses fluorescence-activated nuclei sorting (FANS) to isolate and profile nuclei from multiple different human brain cell-types from frozen post-mortem tissue. Because NeuN is not an optimal marker for neuronal nuclei from fetal cortex, we have optimized a method using a SATB2 antibody to purify nuclei from excitatory neurons in both fetal and postnatal cortex. Purified populations of nuclei are amenable to simultaneous profiling of i) DNA modifications (via bisulfite sequencing / array), ii) histone modifications (via CUT&Tag), iii) open chromatin analysis (via ATAC-seq), and iv) gene expression (via RNA-seq).
Materials

ABC
Supplier Catalogue No
BD FACSAria™ III Cell SorterBD Biosciences 648282-23
Sorvall WX 80+ Ultracentrifuge Thermo Scientific™ 75000080
7mL Dounce Tissue Grinder DWK Life Sciences 357542
PA Thin-walled ultracentrifuge tubesThermo Scientific03699
Table 1: Specifications of the equipment required for FANS protocol


ABC
Supplier Catalogue No
D-Sucrose (Molecular Biology)Fisher Scientific10638403
Calcium chloride (CaCl2) anhydrous, granularSigma-Aldrich C1016-100G
Magnesium acetate ( Mg(Ace)2), 1M aq. solnAlfa AesarJ60041
UltraPure 0.5M EDTA, pH 8.0Invitrogen15575020
Thermo Scientific 1M Tris-HCI Buffer, pH 8.0Fisher Scientific 15568025
1,4-Dithiothreitol (DTT) - crystalline powderSigma-Aldrich 3483-12-3
Triton X-100Sigma-Aldrich T9284
UltraPure DNase/RNase-Free Distilled Water (ddH2O)Fisher Scientific12060346
Bovine Serum Albumin (BSA)Sigma-Aldrich A9647-500G
PBS Phosphate-Buffered Saline (10X) pH 7.4Fisher Scientific10722497
Thermo Scientific™ RiboLock RNase Inhibitor (40 U/µL)Fisher Scientific10389109
TRIzol LS ReagentInvitrogen11588616
BAMBANKER serum free cell freezing mediumBioCat GmbH BB03-NP
BD FACSDiva CS&T Research BeadsBD Biosciences 655051
BD FACS Accudrop BeadsBD Biosciences 345249
BD FACSFlow Sheath Fluid 20LBD Biosciences 342003
BD FACS Clean SolutionBD Biosciences 15875858
BD FACS Rinse SolutionBD Biosciences 340346
Table 2 : Specification of reagents required for FANS protocol


ABC
Lysis Buffer (LB)
StockAmount
0.32M Sucrose              5.47 g     
5mM CaCl21M250 µL   
3mM Mg(Ace)2              1M150 µL    
0.1mM EDTA                   0.5M10 µL  
10mM Tris-HCl, pH 8     1M500 µL    
1mM DTT                        3M17 µL   
0.1% Triton X-100           50 µL
Optional: RiboLock RNase Inhibitor 0.2U/µL40U/ µL5 µL / 1mL
Adjust with ddH2O to  50 mL     
1.8M Sucrose Solution (SS)
1.8M Sucrose                   30.78 g
3mM Mg(Ace)2                  1M150 µL
1mM DTT 3M17 µL
10mM Tris-HCl, pH8         1M 500 µL
Adjust with ddH2O to 50 mL
5% BSA Solution (BB)
BSA                                                           200 mg
1x PBS4 mL
Staining Buffer (SB)
0.5% BSA5% BSA Solution (BB)400 µL
10X PBS400 µL
Optional: RiboLock RNase Inhibitor 0.2U/µL40U/ µL5 µL / 1mL
Adjust with ddH2O to 4 mL  
Table 3 : Recipes for buffers and solutions required

Supplier Thermo Scientific™
Model Sorvall™ WX 80+
Rotor TH-641
Speed 25,200 RPM / 108670.8 x g
Acceleration 9
Deceleration 5
Temperature 4°C
Table 4: Ultracentrifuge specification and conditions

ABCDE
Antibody Preconjugated Supplier Cat No Dilution
Hoechst 33342 -- Abcam ab228551 1:500
Anti-SATB2to Alexa Fluor 488 Abcam ab196316 1:1000
Anti-NeuN to Alexa Fluor488 Millipore MAB377X 1:1000
Table 5: List of antibodies required for FANS protocol

Troubleshooting
Nuclear prep for FACS separation (using SATB2 and Hoechst)
The protocol below yields approximately 660,000 SATB2+ve, 800,000 SATB2 -ve nuclei per Amount200 mg of frozen human fetal post-mortem cortex tissue. Recovery might vary from sample to sample due to high inter-sample variability (developmental stage and density brain collection, cortex sub-areas, fat content of tissue sectioned, and white to grey matter ratio). Yield will also vary for NeuN against developmental age, and we have shown NeuN is not suitable in pre-natal samples, but can be used in post-natal and adult cortex to collect a comparable population.

Refer to Materials-Table 1 for details about the equipment required and to Materials-Table 2 for specifications of reagents required.
Note
Throughout this protocol, SATB2 and NeuN are interchangeable as the neuronal antibody marker, depending on age of the individual being profiled.

Solution and buffer preps

  • Lysis Buffer (LB)
  • Sucrose Solution (SS)
  • Staining Buffer (SB)

Solutions should be kept at Temperature4 °C or TemperatureOn ice . Refer to Materials-Table 3 for recipes of solutions and buffers.
Note
LB and SS can be prepared a week in advance, with DTT added on the day of use. Solutions should be stored atTemperature4 °C once made, preferably in brown glass bottles.


Note

SB should be prepared fresh each day.

Note
Samples are homogenised as bulk tissue using aAmount7 mL Dounce homogeniser and then equally divided into two ultracentrifuge tubes.




Nuclei isolation
1. Pre-cool the ultracentrifuge, including the rotor and swing buckets, toTemperature4 °C Duration00:30:00 before starting this stage of the protocol.
2. All buffers and the Dounce homogenisers should be pre-cooled on ice.
4. Add DTT (Concentration1 millimolar (mM) final concentration ) to the SS and LB (i.e. Amount17 µL 3M DTT per 50 mL of SS/LB)
5. TransferAmount2 mL LB to the homogeniser per Amount200 mg human brain tissue
6. Add the dissected tissue sample into the homogeniser
7. Wait Duration00:05:00 before douncing the tissue to allow the sample to defrost

To reduce heat caused by friction, the Dounce homogenisation step should be performed on ice with gentle strokes, and care should be taken to avoid foaming.

Note
Using the "TIGHT" pestle helps reduce the number of strokes required to reach full tissue disruption.

Note
The number of strokes required to fully homogenise the tissue may vary between samples due to heterogeneity in cellular composition, lipid content, and the amount of connective tissue.

Note
In comparison to adult tissue, the fetal tissue dissociates very easily, with fewer strokes required, and the lysate will be very pale. However, the cells are densely packed in a fetal brain, so we retained the same volume of lysis buffer to perform the dissociation and staining protocols.
Because the fetal brain is more easily dissociated, fewer strokes are required, and we have even seen success with gentle pipetting to agitate the tissue, rather than using a dounce.


Figure 1 Example of adult brain tissue sample
A) only partially homogenised B) complete homogenisation.
Fetal samples will appear paler, with fewer pieces of floating lipid based tissue.
8.TransferAmount8 mL SS (1.8M) to PA thin-walled ultracentrifuge tubes
9.Carefully overlay with tissue homogenate (1 mL per tube) - using a P1000 pipette, releasing slowly down the side of the tube
10.Overlay with another Amount1 mL LB – do not worry about disrupting the homogenate phase
11.Balance opposite tubes by weight with 1x PBS using a fine microbalance
12.Perform ultracentrifugation for Duration00:45:00 (see Table 4 for centrifuge specification and conditions)






1h 20m
After Ultracentrifugation step

  1. Pour off any the supernatant, taking care not to dislodge the pellet (90-degree inclination of the tube). If the pellet is hard to see, it is okay to leave 100-200 µL solution in the ultracentrifuge tube
  2. Re-suspend pellet in SB (Amount750 µL ), gently pipette up and down
  3. Let samples sit on ice for Duration00:15:00 at least (Blocking step)
  4. Transfer volume into 2 mL tubes
  5. Rinse out ultracentrifuge tubes in order to maximise nuclei collection by adding Amount750 µL of SB per tube, pipetting up and down several times, and transferring into the 2 mL tubes
  6. Centrifuge step: for Duration00:05:00 ,Temperature4 °C
  7. Discard supernatant (pipetting off, or pouring off gently)
  8. Re-suspend each nuclei pellet in fresh SB (500 µL)
  9. If the sample was split then pool together pellets from the same sample (Final Volume = Amount1 mL )
  10. Add DNA dye (Hoechst, 1:1000) and mix thoroughly via inversion.
  11. Pipette out Amount100 µL of nuclei solution for the Unstained Control (Hoechst dye only) and transfer to a new 2 mL tube
  12. Bring the volume up to Amount500 µL for the Unstained tube with fresh SB
  13. Replace the 100µL taken from the Stained" tube with 100 µL of fresh SB (Final Volume =Amount1 mL Amount1 mL )


Note
We don't add 1 mL of SB to the ultracentrifuge pellet, as there is always residual liquid remaining, and this would push the volumes above the 2 mL maximum in the 2 mL tubes.


Note
The amount of sample removed for the unstained control can vary, if you're concerned about wasting material. It is only used to establish the gating thresholds, or to collect a non-sorted population.







Immunostaining

  1. Add your antibody of choice (Ab) to staining tube:
SATB2 Alexa488 (1:1000)
or
NeuN Alexa488 (1:1000)

Refer to Table 5 for specifications of the antibodies used

2. Incubate tubes for Duration01:30:00 on the rotor (speed=14 max) at Temperature4 °C , keeping the tubes in the dark
3. Washing step: Centrifigation1000 x g for Duration00:05:00 , Temperature4 °C (both "Stained" and "Unstained" tubes)
4. Discard supernatant (by pipetting off)
5. Re-suspend in fresh SB (Amount1 mL for the Unstained tube, Amount1.5-2 mL for the Stained tube - depending on pellet size)




Fluorescence-Activated Nuclei Sorting (FANS)
5m
For machine start-up, CST and Accudrop calibrations refer to BD FACSAria III User’s Guide for guidance and troubleshooting. The following instructions describe FANS using BD FACSAria III. Other FACS platforms can be used but might require modifications to the protocol.
General Gating Parameters

For each sample, load stained and unstained tubes individually for data acquisition. A preliminary qualitative analysis of the acquired data is essential to select the appropriate gating strategy to maximize the nuclei capture while excluding unnecessary debris and to ensure optimal signal/noise ratio.

Gating Parameters (X-axis:Y-axis):

FSC-A:SSC-A (Size, cell granularity or internal complexity)
SSC-W:SSC-A (to gate out doublets)
FSC-A:DAPI-A (to gate the single nuclei population)
DAPI-A:FITC-A (to gate SATB2 or NeuN stained nuclei)
Note
Refer to Figure 2 for a visualization of the gating strategy. Gating is conserved as much as possible across the age trajectory, but can vary due to developmental changes. It's important to note that the populations should still be present when using SATB2



Figure 2. FANS gating strategy on a fetal sample 13 post conception weeks of development (13 pcw).
(a) Particles smaller than nuclei (black dots) were eliminated with an area plot of forward-scatter (FSC-A) versus side-scatter (SSC-A), with gating for nuclei-sized particles inside the gate (box).
(b) Plots of height versus width in the side scatter channel are used for doublet discrimination with gating to exclude aggregates of two or more nuclei.
(c) Doublet discrimination gating was used to isolate nuclei determined by subgating on Hoechst 33342. (d, e,) Subsequent scatterplots discerning
(d) SATB2-Alexa Fluor488–conjugated antibody staining (purple) for SATB2 stained neuronal cells and SATB2 non-stained nuclei (blue) for non-neuronal cells
(e) SATB2-Alexa Fluor488–conjugated antibody staining (purple) in a contour plot.
Data recording settings

In line with the experiment design, FSC, SSC, DAPI, FITC, are the parameters for which voltage values may need to be slightly adjusted due to experiment/ inter-sample variability.
It is advisable to set the threshold value at 200 during data recording. Moreover, in the acquisition dashboard tab, we recommend setting Events to Record ≤3000, Event to display ≤1000 and Flow Rate = 1.0 (1,000 events per second) in order to increase the accuracy of signal detection.
The flow rate can be increased during sample collection to reduce the sort speed (ideally max events per second =1,500 for a 100-micron nozzle). However higher flow rates impact the data resolution and accuracy of events detection, and subsequent sorting of cellular fractions (see BD FACSAria III User’s Guide for details).
During analysis, recorded data is displayed in plots, while gates are used to define populations of interest for selection. Figure 3 shows a representative example of the two most common outcomes we often observe.

Figure 3. Representative example of inter-individual variability.
The data shown here are derived from two different control prefrontal cortex specimens of comparable age, gender, and brain collection which were processed in parallel following the same procedure.
a) sample from 13pcw, clean nuclei isolation, but with smaller SATB2+ve fraction with clear separation of populations
b) poor sample separation on a 20pcw sample, but contour map demonstrates distinct populations.
Sample Collection

  1. 1. LoBind Tubes (Eppendorf, Cat No:30108051) are required to collect nuclei (to maximize sample recovery of nucleic acids by significantly reducing sample–to–surface binding).
  2. 2. Collected fractions can be used directly for downstream applications (e.g. DNA/RNA extraction, chromatin shearing).
  3. 3. If you are collecting for DNA or RNA, as soon as the number of events desired is reached, transfer the tubes TemperatureOn ice , do not hold them at TemperatureRoom temperature .
Note
During collection, it is crucial to regularly pause the sorting to mix the two phases in order to preserve the integrity of resulting RNA preparations.

Note
For RNA extraction, LoBind Tubes should each contain Amount1 mL of pre-chilled TRIzol™ LS Reagent (Fisher Scientific, Cat No: 11578616) prior to sorting. If possible, use a chilled collection tube holder on your FACS machine.


4. Keep samples TemperatureOn ice and in the dark for the entire duration of the sorting.
5. Lightly vortex sample tubes to make the mixture homogeneous (not clumped) before loading the tube into the FACS chamber.
6. Load the UNSTAINED control tube into the chamber first to postion Unstained gating boxes.
7. Proceed by collecting STAINED tube by simultaneously sorting for SATB2+ve (positive) or SATB2-ve (negative) and proceed with nuclei collection.

Note
We observe great variability of staining profile in the fetal samples across the trajectory of development. We advise you to not expect as conserved patterns as observed in adult cortex.
8. For long term storage of collected nuclei Centrifigation1000 x g, 4°C for Duration00:05:00
9. Carefully remove supernatant.
10. Add Amount100 µL BAMBANKER to the tube.
11. Gently resuspend.
12. Store in Temperature-80 °C freezer

5m
General Recommendations for the user

For every new experiment we recommend performing the following steps:

When loading your tube into the FACS machine, run the unstained / IgG control sample first as this aids in setting the baseline parameters
Check your event rate in the Acquisition Dashboard window. If it is greater than 1500 evt/s turn down the “flow rate” or unload and dilute the sample further. If less than 100 evt/s, turn up the “flow rate” (don’t exceed a flow rate of 5.0 if possible, as the instrument is less focused and more inaccurate at higher flow rates)
In the Acquisition Dashboard window choose the appropriate “stopping gate” and “storage gate” (when working with nuclei, set as “Nuclei” and “All events” respectively)
Choose the range of “events to record” and “events to display” that best suits your purpose (≥ 5000 for both is advised)
Under the “threshold” tab in the Cytometer window, change the threshold (should be set for FSC) so that any small events in the bottom corner of the FSC vs SSC graph (caused by general cell debris and dust) are no longer shown. The threshold should not be set too high so that it causes an arbitrary, artificial cut-off through the left side of your population but not so low that small events caused by debris/dust are visible (ideally between a threshold 200-500).
Under the “parameters” tab in the Cytometer window, adjust the “FSC” and “SSC” values to get your population sitting in the centre of the FSC vs SSC graph (a re-adjustment of the “threshold” may be required at this point). It is essential to select “restart” each time any of the parameters are changed to update the events being displayed to ensure only events are recorded under the new settings.
Adjust or draw a new gate in the FSC vs SSC plot to encompass the population of interest.
Look in the scatter graph of SSC-A vs SSC-W (if you opened a blank experiment you will need to draw one). Right-click on the graph and check it is only displaying the events encompassed by your previous FSC vs SSC gate. Adjust or draw a gate for SSC A vs SSC W to encompass all of the main population to the left of the graph and exclude outliers to the right (these are doublets and other cell debris clumps)
Under the “parameters” tab in the Cytometer window adjust parameters for the fluorochromes selected so the unstained / IgG control sample sits close to 0 for the fluorochrome on a graph of FSC vs fluorochrome.
Load the stained samples and check the stained population has a clear increase in signal for the fluorochrome in comparison to the unstained (signal generally should not exceed 10x4). Several minor re-adjustments of the fluorochrome’s “parameters" may be necessary for the stained sample at this stage. If so, the unstained / IgG control has to be reset and re-recorded.
Note
WARNING – Do not change parameter settings between samples you wish to compare, if you do you will need to re-record all samples using the changed parameters.

11. Select the correct option for the collection device in the Sort Layout window ( we recommend “4-Way Purity” for general collection)
12. Regularly check your “Efficiency” in the Sort Layout window value. Between 80-100% is ideal, 70% is acceptable if less than 70% either the sample is too concentrated or you are sorting a rare population. Although the “flow rate” in the Acquisition Dashboard window can be increased to make the sort quicker, faster flow rates will negatively impact the efficiency.
13. Check the “Electronic abort rate” (N° errors /sec) and “Electronic abort count” (Tot N° of errors) at the bottom of the Acquisition Dashboard window. These parameters measure potential miss-sorts (different from efficiency as efficiency measures undetermined drops which are directed to the “Waste” and therefore lost but do not contaminate).“Electronic abort rate” should be <1% of total events per second.
14. For long sorts, gate positions should be regularly monitored, especially for stained populations as fluorochromes lose intensity over time and the population can shift towards the unstained. Gates can be moved during long sorts to compensate.