Single-nucleus RNA-seq data generation

Nuclei preparation from frozen tissues for snRNA-seq using grinding
 

A. Reagents and consumables:

Prepare buffers fresh and leave on ice.

• DRAQ7 (Cell Signaling Technology, #7406S) or other nucleardye.
• Sony Cell Sorter 100 μM Chip (Sony, LE-B3001)
• Sony Cell Sorter (Sony, SH800S)
• Eppendorf centrifuge (Eppendorf, 5920R) or other swinging bucket centrifuge.
• 30 μM CellTrics (Sysmex, 04-0042-2316)

B. Tissue pulverization

Ensure the samples are kept frozen on dry ice throughout pulverization.

1. Pour liquid nitrogen into a mortar and pestle until frozen.
2. Remove tissue from tube and place into liquid nitrogen with a clean cold spatula.
3. Grind up the sample with liquid nitrogen using the mortar and pestle.
4. Use a clean cold spatula to scoop ground tissue back to the original tube.
5. Place the sample back onto dry ice or directly to -80 °C.
6. Clean the mortar and pestle with 10% bleach and 70% ethanol before using it for the next sample.

C. Nuclei preparation

1. Prechill any tubes and buffers. Set the centrifuge to 4˚C.
2. Transfer pulverized tissue from -80 to dry ice; freeze spatula and 1.5 ml 1.5 mL Lobind tube (Eppendorf) in liquid nitrogen.
3. Transfer 30-40 mg of tissue into frozen 1.5 ml Lobind tube (Eppendorf) and immediately add 500 ul LB, pipette 10x up and down and place on ice.
4. Incubate for 5 minutes in cold room on overhead rotator.
5. Centrifuge nuclei suspension (500 rcf, 5 min, 4˚C).
6. Aspirate supernatant and discard.Resuspend nuclei in 400 μl of SB by mixingwith a p1000 pipette 10 times.
7. Filter nuclei by passing lysate through a 30 μM CellTrics filter into a new 1.5 ml Lobind tube. Wash filter and tube with an additional 400 μl of SB.
8. Add 6 ul of DRAQ7 and pipette to mix with p1000 5 times. Incubate on ice for 10 minutes.
9. Sort 60,000-75,000 nuclei into a Lobind tube containing 50 μl of collection buffer.
10. Centrifuge nuclei (1000 rcf, 15 min, 4˚C).
11. A tiny blue pelletshould be visible,indicating nuclei. Aspiratesupernatant and discard. Resuspend pellet in 35 μl of reaction buffer.

D. Nuclei counting

Reagents:

• Trypan Blue Solution, 0.4% (Thermo-Fischer, 15250061)
• Hemocytometer (Thermo-Fischer, #0267151B)

1. Ensure that nuclei mix is homogenous by resuspending gentlywith a p200 pipette 5 times.
2. Combine a 5 μl aliquot with 5 μl of Trypan blue and mix with a p20 pipette (set to 10 μl) gently 5 times.
3. Dispense on the hemocytometer and count nuclei. Take pictures to record and assess nuclei (e.g. are the nuclei too “ragged” looking at the edges = poor nuclei)
4. Use a volume equal to 12,000 nuclei of nuclei suspension to load onto the Chromium Controller (10x Genomics).

E. 10x single-nucleus RNA-seq kit instructions

Prepare GEM reactions and libraries using the Chromium Next GEM Single Cell 3ʹ Reagent Kits v3 or v3.1 kit (10x Genomics) as per manufacturer specifications with the following PCR cycle numbers:

• 12 PCR cycles for cDNA preparation.
• 10 PCR cycles for Library Amplification SI-PCR