TITLE: Nuclei Prep from Frozen Mouse Brain Tissues with Optional Sucrose Column for single nuclei RNA-seq
AUTHORS: Greggory  A Perry, Sandra L Daigle, Paul  Gabriel, Diane  Luo, Jessica  Grassmann, William F F Flynn, Elise T Courtois, Paul  Robson

[DESCRIPTION]
This SOP aims to extract nuclei from unfixed snap-frozen mouse prefrontal cortex, hippocampus, and Striatum tissues (~3-15mg). It was optimized and tested on mice ranging in age groups from 3, 6, 14, and 15-month-old mice on different diets, including high-fat (45%) and regular chow (6%). Other differences included sex, strain, and genotype. These preps resulted in a minimum of debris for downstream snRNAseq HT assays on the 10X Genomics chromium X controller (HT, High Throughput chips). 

This protocol was also used to perform snRNAseq assays from human glioblastoma snap frozen tissues. Follow BSL-2 guidelines when working with human tissues.  These preps resulted in a minimum of debris for downstream snRNAseq ST assays on the 10X Genomics chromium X controller (ST, Standard Throughput chips). 

This protocol enables to process up to 8 samples simultaneously. 

[BEFORE_START]
A. Pre-cool centrifuge, buffers, and consumables with sample contact (e.g., gentleMACS C Tube and Strainers) at 4 °C.
B. Per extraction, add 0.2U/ul RNase inhibitor to one 1.6 ml aliquot of Miltenyi Nuclei Extraction Buffer (8µl of 40U/uL RNase inhibitor) and keep on ice.
C. Per extraction, add 1 mM DTT to one 750 µl aliquot of Sucrose Solution (0.58µl 1.3M DTT) in a 2 ml tube and keep on ice.
D. Per extraction, prepare 750ul Nuclei Suspension Buffer per sample.

[GUIDELINES]
A. Use wide-bore pipette tips to avoid damage when handling tissue or nuclei, and pipette slowly.

B. Keep tissue on dry ice until ready to start gentleMACS.

C. Miltenyi gentleMACS Recommendations.
Tissue 1-4 mg - 4C_nuclei_1
Tissue 5-7 mg - cus_4C_nuclei_5
Tissue 7-15 mg - cus_4C_nuclei_10 

The "cus" protocols are custom SOP from Miltenyi. It adds 5 or 10 minutes of mixing at 20 RPM after the Miltenyi 5-minute disassociation SOP 4C_nuclei. 
    * Optimization may be needed depending on tissue type, size, and condition.

D. "To sucrose" or "not to sucrose".
If initial sample size is < 5 mg, it is recommended to wash nuclei without using a sucrose gradient (Step 3).  If initial sample size is > 5 mg, it is recommended to purify nuclei using a sucrose gradient (Step 2).

E. Sucrose column recommendations.
After the sucrose cleanup, a tissue less than 3-5 mg may have a very low nuclei yield. It is recommended to test this first on a representative sample to ensure precious samples are not lost. 

Hoechst/Syto RNA Select Staining Guidelines

1. Extracellular debris should be minimal after the nuclei have been passed through the sucrose column.
2. Extracellular debris will be present in a much greater quantity if a sucrose column is not used. Debris can interfere with downstream 10x operations by introducing clogs and background ambient RNA.  
3. Nuclei should be intact, with minimal blebbing and clumping. Blebbing of nuclei will make them more prone to clumping, which can introduce clogs when loading the 10x chip. 
4. The Hoechst and Syto Select staining should be uniform through the nuclei, but the Syto staining usually has more variation.

If working with human tissue, follow all institutional rules and guidelines.

[STEPS]
SECTION: Procedure- gentleMAC
1. 
Miltenyi gentleMACS Procedure for Tissue Lysis and nuclei extraction

Please see the Guidelines & Warnings section before beginning. This protocol was optimized to process up to 8 samples simultaneously. Some practice is required to process 8 samples at once. Work fast but cautiously.. 

  

SECTION: Sucrose Column
2. Sucrose Column Purification
SECTION: Wash without Sucrose Column
3. 
Washing nuclei without the use of a sucrose column 
SECTION: Procedure- gentleMAC
1.2. Transfer frozen tissue piece(s) into the gentleMACS C Tube containing lysis buffer and directly proceed with the following steps until samples are dissociated. 

Note: Do not let frozen samples thaw before dissociation, as endogenous RNase might degrade RNA. 
SECTION: Procedure- gentleMAC
1.1. Add 1250 µL on ice  to each pre-cooled gentleMACS C Tube. 

SECTION: Procedure- gentleMAC
1.4. Run gentleMACS Program based on tissue size on the gentleMACS Dissociator at 4 °C.
See note C under Guidelines & Warnings.
SECTION: Procedure- gentleMAC
1.3. Close gentleMACS C Tube and place it on the gentleMACS Dissociator.

Be sure the cap is closed completely.

  
 

SECTION: Procedure- gentleMAC
1.5. A quick spin in a swinging bucket at 500 x g, 4 °C for 10-15 sec may be necessary.

  

SECTION: Procedure- gentleMAC
1.8. Remove supernatant and resuspend nuclei in 500 µL
    
If the pellet is loose, leave 50 µL in the tube.
    
See the Guidelines & Warning section D to decide to proceed to step 2 (sucrose column) or 3 (no sucrose column) next.

SECTION: Procedure- gentleMAC
1.6. Pipette mix with wide bore tip and filter through pre-wet 100µm filter into a cold 2mL tube.
Pre-wet filter with Nuclei Extraction Buffer.
Add 250 µL to rinse the filter.
Spin in a benchtop centrifuge for 5-10 seconds to remove foam if necessary.

  
SECTION: Procedure- gentleMAC
1.7. Spin down nuclei, 500 x g, 5 min, 4 °C in a swinging bucket.

 

SECTION: Sucrose Column
2.1. In a 2mL microcentrifuge tube with 750 µL, layer 500 µL on top, forming two layers.

 

SECTION: Sucrose Column
2.6. Pre-wet 40μm filter with Nuclei Suspension Buffer.

Gently pipette mix 10-15x with wide bore tip and filter through pre-wet 40μm filter into new cold 1.5mL tube. 

Be careful not to wash down sucrose waste from the tube walls.

Proceed to Step 4 and 5.
    
SECTION: Sucrose Column
2.5. 
Resuspend the nuclei pellet in 50-150µl Nuclei Suspension Buffer and let sit on ice without pipetting for 3-5 minutes to loosen the pellet.
SECTION: Sucrose Column
2.4. Carefully pour off and blot the tube or aspirate the debris with the supernatant. Remove as much supernatant as possible without disturbing the nuclei pellet.
SECTION: Sucrose Column
2.3. After centrifugation, purified nuclei should be visible as a pellet on the bottom of the tube, and white matter/debris should float midway in the 2 ml tube.

   
SECTION: Sucrose Column
2.2. Spindown, 16000 x g, 15 min, 4 °C in a fixed angle rotor.

SECTION: Wash without Sucrose Column
3.1. Add 500 µL and gently pipet mix 5-10x using wide bore tip to suspend nuclei.

SECTION: Wash without Sucrose Column
3.6. Pre-wet 40μm filter with Nuclei Suspension Buffer.

Gently pipette mix 10-15x with wide bore tip and filter through a pre-wet 40μm filter into new cold 1.5mL tube.
Proceed to step 4 and 5.
SECTION: Wash without Sucrose Column
3.5. Resuspend the nuclei pellet in 50-150µl Nuclei Suspension Buffer and let sit on ice without pipetting for 3-5 minutes to loosen the pellet.
SECTION: Wash without Sucrose Column
3.4. Repeat Steps 3.1 - 3.3 for a total of 2 washes.
SECTION: Wash without Sucrose Column
3.2. Centrifuge in a swinging bucket 350 x g, 5 min, 4 °C.

SECTION: Nuclei counting
4. Nuclei counting with Trypan Blue / AOPI stain

Obtain nuclei in suspension buffer from steps 2 or 3.

Proceed to Step 4.1 if using Luna FX7 cell counter and AOPI stain to count nuclei.

Proceed to Step 4.2 if using Countess FL II cell counter and Trypan Blue stain to count nuclei.

Proceed to Step 4.3 if using Nexcelom Cellometer K2 and AOPI stain to count nuclei.

Note: Trypan Blue staining is useful to visualize debris and nuclei qualitatively.  The counts using Trypan include both nuclei and debris.  AOPI is used to count only nuclei.  By comparing Trypan and AOPI counts, one can quantitatively asses the amount of debris present in samples preps.
SECTION: Loading nuclei on 10x Genomics chip
6. 10x Genomics downstream applications

Follow 10x recommendations for your specific needs. 


For a 10x standard chip (ST) and High Throughput (HT) chip, calculate the number of nuclei that needs to be loaded for each channel, and according to the experimental design.

    Loading examples:

    ST - Targeting 6,000 nuclei
                    - load 12,000 to recover 12,000 taking into account a 50% capture rate.

    HT - Targeting 20,000 nuclei
                    - load 33,300 to recover 20,000 taking into account a 60% capture rate.

    For more detailed guidelines visit https://www.10xgenomics.com/ for more details.
SECTION: Nuclei counting
4.1. Nuclei counting with LunaFX7

A. Add 1uL AOPI stain and 9uL suspended nuclei to a .5 ml microcentrifuge tube or PCR strip tube and mix gently by pipetting 5x. 

Examples:
1ul AOPI/9ul sample
1ul AOPI/4.5ul sample/4.5 ul suspension buffer 

B. Turn on Luna FX7 cell counter. 
Select "fluorescence cell counting" and "cell lines & primary cells" with the touch screen menu.

C. Select the settings tab and choose the correct slide and wells being counted.

D. Load preferred protocol for fluorescence cell counting.  

E. Load preferred protocol for fluorescence cell counting.  

F. Select Count, insert the slide into the LunaFX7, and pre Count on the right-hand menu.

G. Record desired data after Count is finished using the save and print command, which allows renaming of the sample, or use quick save, which auto-generated a name.

H. Below is an example of Luna FX7 images. 

  


SECTION: Nuclei counting
4.2. Nuclei counting with Countess FL II

A. Add 6 uL Trypan Blue stain 0.4% and 6 uL nuclei suspension to 0.2 mL PCR strip tube.  Mix gently by pipetting 5-10x.

B. Load 10 uL into Countess cell counting chamber slide.

C. Visually assess nuclei sample in slide using bench top microscope, 20x magnification.  Note debris present, if any, and overall nuclei shape/integrity.

D. Load slide into Countess FL II.  Adjust brightness and focus if necessary.  Click "Count".

E. Once counting has been completed, record nuclei concentration, viability, and size.  Save data with correct date and sample name.

F. Below is an example of Countess FL II readout showing "dead" cells, or nuclei.
  

SECTION: Nuclei quality QC: Hoechst and SYTO RNASelect staining of nuclei
5. Hoechst and SYTO RNASelect staining of nuclei

Keep all stains and dilutions on ice in the dark  4 °C.

Choose 5.1 for Leica DM4 B or 5.2 for PerkinElmer Opera Phenix.
SECTION: Nuclei quality QC: Hoechst and SYTO RNASelect staining of nuclei
5.1. Nuclei QC with Leica DM4 B

Imaging:
Microscope - Leica DM4 B
Camera - Leica DCF7000 GT

Exposure time:
Hoechst - 45 ms, gain 5.5
SYTO RNASelect - 800 ms, gain 3.4
Brightfield - 30 ms, gain 8.8

Magnification - 40X

 The exposure time and gain may need to be adjusted depending on the microscope used.

A. Make a 1:80 dilution of Hoechst stain (1ul into 79ul PBS)
B. Make a 1:500 dilution of SYTO RNASelect stain (1ul into 499ul PBS)

C. Mix well and aliquot 18ul of nuclei suspension into a new microcentrifuge tube. Dilute if needed at this point, but the total volume should be 18ul.

D. Add 1ul 1:80 Hoechst stain and 1ul 1:500 SYTO RNASelect stain to 18ul suspended nuclei and mix gently 5x pipetting. Immediately place the mixture in a light-blocking box and place it at  20 min, 4 °C.

Expected results of nuclei put through a sucrose column are shown below.

See guidelines for further details.


   
SECTION: Nuclei quality QC: Hoechst and SYTO RNASelect staining of nuclei
5.2. Nuclei QC with Opera Phenix confocal microscope with 40x water objective. 

A. Dilute SYTO RNASelect stain 1:1000 and dilute Hoechst 1:100 in DPBS.  Keep dye dilutions on ice and protected from light.

B. Place a 384-well plate (PerkinElmer CellCarrier Ultra) on ice.

C. Add 20 uL of nuclei suspension to a 0.2 mL strip tube.

D. Add 1 uL each of diluted SYTO RNASelect and Hoechst stains into tube containing nuclei suspension.  Pipet mix 5-10x.

E. Transfer contents of strip tube into 384-well plate.  Incubate 384-well plate at   20 min, 4 °C protected from light.

F. Image 384-well plate using the following parameters, ensuring that no channels are over/underexposed:
    Plate Type: 384 PerkinElmer CellCarrier Ultra
    Autofocus: Two Peak (Default)
    Objective: 40x Water, NA 1.1
    Opt. Mode: Confocal
    Binning: 2
    Channel Selection:
        Brightfield
            Exposure Time = 200 ms
            Power = 100%
        SYTO RNA Select
            Exposure Time = 100 ms
            Power = 50%
        HOECHST 33342
            Exposure Time = 100 ms
            Power = 50%
    Layout Selection (Well)
        Number of Fields = 49
        Overlap = 5%
    Layout Selection (Stack)
        First Plate =  -1.0 um
        Number of Planes = 6
        Distance = 3.0 um
        Last Plane = 14.0 um
        Overall Height = 15.0 um

G. Example nuclei QC images are below.
    
    

SECTION: Wash without Sucrose Column
3.3. Remove supernatant, being careful not to disturb the nuclei pellet.
SECTION: Nuclei counting
4.3. Nuclei counting with Nexcelom Cellometer K2

A. Add 11 uL AOPI stain and 11 uL nuclei suspension to 0.2 mL PCR strip tube.  Mix gently by pipetting 5-10x.

B. Load 20 uL into Cellometer SD100 slide.

C. Record nuclei concentration, viability, and size.