Nuclei Prep from Frozen Mouse Brain Tissues with Optional Sucrose Column for single nuclei RNA-seq

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..

Add 1250µL On ice to each pre-cooled gentleMACS C Tube.

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.

Close gentleMACS C Tube and place it on the gentleMACS Dissociator.

Be sure the cap is closed completely.

Equipment

Value	Label
gentleMACS	NAME
gentleMACS™ Octo Dissociator with Heaters	TYPE
Miltenyi Biotec	BRAND
130-096-427	SKU

Run gentleMACS Program based on tissue size on the gentleMACS Dissociator at 4°C.

See note C under Guidelines & Warnings.

A quick spin in a swinging bucket at 500x g,4°C,0h 0m 0s for 10-15 sec may be necessary.

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 250µL to rinse the filter.
• Spin in a benchtop centrifuge for 5-10 seconds to remove foam if necessary.

Spin down nuclei, 500x g,4°C in a swinging bucket.

Remove supernatant and resuspend nuclei in 500µL

If the pellet is loose , leave in the tube 50µL in the tube.

See the Guidelines & Warning section D to decide to proceed to step 2 (sucrose column) or 3 (no sucrose column) next.

Sucrose Column Purification

In a 2mL microcentrifuge tube with 750µL, layer 500µL on top, forming two layers.

Spindown, 16000x g,4°C in a fixed angle rotor.

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.

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.

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.

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.

Washing nuclei without the use of a sucrose column

Add 500µL and gently pipet mix 5-10x using wide bore tip to suspend nuclei.

Centrifuge in a swinging bucket 350x g,4°C.

Remove supernatant, being careful not to disturb the nuclei pellet.

Repeat Steps 3.1 - 3.3 for a total of 2 washes.

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.

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.

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.

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.

<img src="https://static.yanyin.tech/literature_test/protocol_io_true/protocols.io.81wgb657ylpk/image%20%281%29.png" alt="Left image shows "dead" cells or nuclei (red, nuclei stained with PI).Right image shows the brightfield for the same field." loading="lazy" title="Left image shows "dead" cells or nuclei (red, nuclei stained with PI).Right image shows the brightfield for the same field."/>

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.

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.

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.

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 ,4°C.

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

See guidelines for further details.

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 ,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.

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/](https://www.10xgenomics.com) for more details.