TITLE: Fluorescence-activated nuclei sorting (FANS) on human post-mortem cortex tissue enabling the isolation of distinct neural cell populations for multiple omic profiling
AUTHORS: Stefania S Policicchio, Barry Chioza, Jonathan P Davies, Joe Burrage, Jonathan Mill, Emma L Dempster

[DESCRIPTION]
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. 
As 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. Furthermore, the valuable nature of human post-mortem tissue means it is important to use methods that maximize the amount of genomic data generated on each sample. 
This protocol describes a method that uses fluorescence-activated nuclei sorting (FANS) to isolate and profile nuclei from multiple different human brain cell-types from frozen post-mortem tissue. 
This protocol can be used to robustly purify populations of neuronal (NeuN+ve), oligodendrocytes (SOX10+ve), microglia (IRF8+ve) and other glial origin nuclei (NeuN-ve/SOX10-ve/IRF8-ve) from adult post-mortem frozen brain, with each tissue sample yielding purified populations of nuclei amenable to simultaneous analysis of i) DNA modifications (via bisulfite sequencing / array), ii) histone modifications (CUT&Tag), iii) open chromatin analysis (ATAC-seq), and iv) gene expression (snRNA-seq).      

[GUIDELINES]
Prior to attempting this protocol please obtain the approval of your Institutional Review Board (IRB) or equivalent ethics committee(s).

[STEPS]
SECTION: Nuclear prep for FACS separation (using SOX10, IRF8, NeuN and Hoechst)
1. The protocol below yields at least 1,000,000 NeuN +ve, 1,000,000 SOX10 +ve, 400,000 IRF8 +ve (when the population is present) and 200,000 triple negative (NeuN-ve/SOX10-ve/IRF8-ve) nuclei per 500 mg of frozen human post-mortem cortex tissue. Recovery might vary from sample to sample due to high inter-sample variability (brain collection, cortex sub-areas, fat content of tissue sectioned, and white to grey matter ratio)

Refer to Materials-Table 1  for details about the equipment required and to Materials-Table 2  for specifications of reagents required. 
1. 
SECTION: Nuclear prep for FACS separation (using SOX10, IRF8, NeuN and Hoechst)
1.1. Solution and buffer preps 

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

Solutions should be kept at 4 °C  or on ice. Refer to Materials-Table  3  for  recipes of solutions and buffers. 
 

 

 


SECTION: Fluorescence-Activated Nuclei Sorting (FANS)
2. 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.
SECTION: Fluorescence-Activated Nuclei Sorting (FANS)
2.1. General Gating Parameters

For each sample, load stained and unstained tubes individually for data acquisition. A preliminary qualitative analysis of the data acquired 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 NeuN stained nuclei)
DAPI-A:PE-A (to gate SOX10 stained nuclei)
DAPI-A:APC-A (to gate IRF8 stained nuclei)
FITC-A: PE-A (to visualize the distribution of triple staining)
 

 

2.2. Data recording settings

In line with the experiment design, FSC, SSC, DAPI, FITC, APC and PE 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 between 200 and 500 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.


  


SECTION: General Recommendations for the user
3. 
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 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.
 

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 are less efficient.
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.

SECTION: Nuclear prep for FACS separation (using SOX10, IRF8, NeuN and Hoechst)
1.2. Nuclei isolation
Pre-cool the ultracentrifuge to4 °C 30 min before starting this stage of the protocol.
All buffers and the Dounce homogenisers should be pre-cooled on ice.
Add DTT (1 millimolar (mM) final concentration ) to the SS and LB (i.e. Amount17 µL 3M DTT per 50 mL of SS/LB)
Transfer 3 mL LB to the homogeniser per 500 mg human brain tissue.
Add the dissected tissue sample into the homogeniser.
Wait 5 min 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.

 

 


 
7.  Transfer 8 mL SS to PA thin-walled ultracentrifuge tubes.
8.  Carefully overlay with tissue homogenate (1 mLper tube) - using a P1000 pipette, releasing slowly down the side of the tube.
9.  Overlay with another1 mL LB – do not worry about disrupting the  homogenate phase
10 .Balance opposite tubes by weight with LB using a fine microbalance.
11. Perform ultracentrifugation for 45 min(see Table 4 for centrifuge specification and conditions).













SECTION: Nuclear prep for FACS separation (using SOX10, IRF8, NeuN and Hoechst)
1.3. After Ultracentrifugation step 
1.  Pour off any remaining 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  solution in the ultracentrifuge tube.
2.  Re-suspend pellet in 1 mL  SB and gently pipette up and down.
3. Let samples sit onon icefor at least  15 min

Blocking step
4.  Transfer volume into 2 mL Lo-Bind Eppendorf tubes.
5.  Rinse out ultracentrifuge tubes in order to maximise nuclei collection by adding SB to each      ultracentrifuge tube (1 mL ) pipetting up and down several times and transferring to the new 2 mL tubes.

Washing step: 
6. Centifuge tubes at1000 x gfor 5 min, 4 °C
7.  Discard supernatant (pipetting off gently).
8.  Re-suspend each nuclei pellet in fresh SB ( 500 µL  ) 
If the sample was split then pool together pellets from the same sample (Final Volume =1.5 mL)
9.   Add DNA dye (Hoechst, 1:1000) and mix thoroughly via inversion.
10. Pipette out 200 µLof nuclei solution for the Unstained Control (Hoechst dye only) and transfer to a new 2 mL tube
11.  Bring the volume up to 1 mL for the Unstained tube with fresh SB.
12.  Replace the volume taken from the Stained" tube with 200 µLof fresh SB  (Final Volume  1.5 mL).


SECTION: Nuclear prep for FACS separation (using SOX10, IRF8, NeuN and Hoechst)
1.4. Immunostaining

1.  Add the following three antibodies (Ab) to Stained tube (1.5ml): 
       
       SOX10 pre-conjugated antibody (1:20 dilution) – [75 µL Ab]
       NeuN Alexa488 (1:1000) – [1.5 µL Ab ]
       IRF8 pre-conjugated antibody (1:150) - [10 µL Ab]

Refer to Table 5  for specifications of the antibodies used 

2. Incubate tubes for at least 90 min on the rotor mixer (speed=11) at 4 °C, keeping the tubes in the dark
3. Washing step: 1000 x gfor 5 min, 4 °C (both "Stained" and "Unstained" tubes)
4. Discard supernatant (by aspiration)
5. Re-suspend in fresh SB (0.5 mL for the Unstained tube, -2 mL for the Stained tube - depending on pellet size)






2.3. Sample Collection

DNA 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).
Collected fractions can be used directly for downstream applications (e.g. DNA/RNA extraction, ATAC-seq, etc) or stored in  -80 °C freezer.          
If you are collecting for DNA or RNA, as soon as the number of events desired is reached, transfer the tubes on ice, do not hold them at Room temperature 
 

 

4. Keep samples on ice 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 and proceed with nuclei collection. 
7. Proceed by collecting STAINED tube by simultaneously sorting for NeuN+ve, SOX10+ve , IRF8+ve  or Triple Negative.  
 

 
8.  For long term storage of collected nuclei 1000 x g, 4 °C  for 5 min   
9.  Carefully remove supernatant.
10. Add 100 µL per 100,000 nuclei collected to the tube.
11. Gently resuspend.
12. Store in -80 °C freezer