Isolation of Nuclei from Flash-Frozen Liver Tissue for Single-Cell Multiomics

All animal experiments were carried out in compliance with German Animal Welfare Legislation and the regulations of the Government of Upper Bavaria. Animal housing was approved according to §11 of the German Animal Welfare Act and performed in accordance with Directive 2010/63/EU.

1. Tissue preparation

1. Sacrifice a 3 month old to 22 month old male C57BL/6J mouse by cervical dislocation. Lay the animal on a dissecting board, secure the extremities with pins, and disinfect the abdomen with 70% ethanol.
2. Perform necropsy as recommended by Treuting et al.¹⁶.
   1. Open the abdomen up to the ribcage, visualize the liver, and use forceps to carefully remove the liver without piercing the lobules.
   2. Extract the intact liver by holding the diaphragm with forceps and removing the connecting tissue with scissors.
3. Wash the organ in cold phosphate-buffered saline (PBS), pat it dry on a clean paper towel, and cut the liver lobules into several pieces for different purposes: FF for single-nucleus isolation and multiomics; paraformaldehyde-fixed (10% PFA) for paraffin embedding; and/or embedded in optimal cutting temperature (OCT) compound for further histological analyses (Figure 1A).
4. Aliquot the liver pieces into cryovials or 5 mL screw-cap tubes, and immediately flash-freeze in liquid nitrogen. Store the frozen liver samples at −80 °C for downstream single-nucleus multiomics experiments (Figure 1B, C).
   ​NOTE: The cryopreserved tissue can be safely stored at −80 °C for several years and should always be transported on dry ice at −80 °C prior to usage.

2. Nuclei isolation

1. Benchtop cleaning and preparation of the buffers and consumables
   1. Clean the working benchtops and pipettes with 70% ethanol and RNase decontamination solution, or use dedicated RNase-free benchtops and materials.
   2. Prechill both the swinging bucket and fixed-angle centrifuges, 1.5 mL/2 mL tubes, and multi-well plates to 4 °C, as detailed below.
   3. Prechill the RNase-free, single-use tweezers used for tissue handling, a disposable sterile scalpel, and a Petri dish on dry ice (−60 °C).
   4. Prechill the Dounce glass homogenizer and pestles on ice (4 °C). Place each pestle (A and B) into a 5 mL tube to avoid direct contact with the ice and potential RNase contamination.
   5. Prepare a diluent solution of iodixanol medium (IDM) (Table 1A), and use it to make 50% and 29% dilutions of the 60% iodixanol stock density gradient medium (Table 1B and Table 1C, respectively).
   6. Prechill all the tubes. For each sample that is going to be processed, prepare the following tubes:
      1. Prepare three 1.5 mL low-DNA binding tubes (one for the filtered tissue homogenate, a second one containing 250 µL of a 50% dilution of iodixanol solution, and a third one for the clean nuclei suspension).
      2. Prepare one 2 mL round-bottom tube containing 500 µL of a 29% dilution of iodixanol solution for the density gradient separation.
      3. Prepare one 15 mL conical tube for the nuclei isolation medium-2 (NIM-2) and homogenization buffer (HB).
   7. Prepare the nuclei isolation medium-1 (NIM-1) (Table 1D), and use it to make the NIM-2 (Table 1E) and, subsequently, the HB (Table 1F). Add both the RNAse inhibitors just before use, as described below in the protocol.
   8. Prepare the nuclei storage buffer (NSB) as described in Table 1G. Add recombinant RNAse inhibitor just before use. Add protein-based RNAse inhibitor into the NSB before use for FACS sorting (optional).
   9. Prepare a 500 mL beaker with sterile water to soak the Dounce homogenizers and pestles following the tissue homogenization for the optimal cleaning and maintenance of the homogenizers.
2. Tissue homogenization
   1. Cut a 20-30 mg (or 5 mm³) tissue piece with a prechilled scalpel inside the Petri dish on dry ice. Then, immediately transfer the Petri dish to wet ice (4 °C). Add 1 mL of HB, and using the cold scalpel, mince the tissue as much as possible to allow it to be easily aspirated with a 1 mL wide-orifice tip.
      NOTE: Always use wide-orifice tips for all tissue/nuclei transfers. As an alternative, 1 mL tips can be cut with a sterile scalpel on a sterile plastic cover to generate wide orifices (Figure 2A).
   2. Collect the tissue suspension, and transfer it to a prechilled 2 mL glass Dounce homogenizer (Figure 2B).
   3. Wash the Petri dish with an additional 0.5-1 mL of HB, and collect all the remaining tissue pieces while keeping everything on ice.
   4. Slowly and carefully do five strokes with loose pestle A on ice. Avoid creating bubbles by using twisting motions of the pestle when pulling it up and down. Ensure that the pestle moves carefully from the top to the bottom of the homogenizer with each stroke.
   5. Subsequently, perform 10-15 slow strokes with tight pestle B on ice. Avoid creating bubbles.
      NOTE: It is recommended to visually inspect the nuclei under the microscope after 10 strokes with pestle B to ascertain if more strokes are needed. Do this by using trypan blue staining (1:1 ratio of trypan to sample) and a manual hemocytometer (e.g., mix 10 μL of trypan blue with 10 μL of nuclei suspension, and use 10 μL of the mixture for examination under the microscope; Figure 2C).
   6. Filter the homogenate through a 50 µm cell strainer while transferring it into a prechilled 1.5 mL tube. Use more than one filter and/or tube for homogenates containing a high amount of connective tissue clumps.
   7. Rinse the homogenizer and the filters used with an additional 0.5-1 mL of HB to thoroughly collect all the tissue homogenate. Proceed to the density gradient centrifugation.
3. Density gradient centrifugation
   1. Centrifuge the filtered homogenate in a prechilled, fixed-angle centrifuge at 1,000 × g for 8 min at 4 °C.
   2. While the sample is spinning, prepare a 1.5 mL tube containing 250 µL of 50% iodixanol dilution and one 2 mL tube containing 500 µL of 29% iodixanol dilution. Keep both tubes on ice.
   3. Following centrifugation, aspirate the supernatant without disturbing the pellet using a vacuum pump.
      NOTE: Using manual pipetting will compromise the quality of the final nuclei suspension.
   4. Add 250 µL of HB to the pellet using the 1 mL wide-orifice pipette tip, and resuspend very slowly.
   5. Transfer 250 µL of the nuclei suspension into a prechilled 1.5 mL tube containing 250 µL of 50% iodixanol dilution, and mix gently but thoroughly to generate a 25% iodixanol/nuclei suspension.
   6. Transfer 500 µL of the 25% iodixanol/nuclei suspension into a prechilled 2 mL tube containing 500 µL of 29% iodixanol dilution.
      NOTE: The 500 µL of the 25% iodixanol/nuclei suspension should be deposited gently on top of the 500 µL of 29% iodixanol solution such that the 25%/29% iodixanol mixtures show clear phase separation. Use the side of the tube wall with the pipette tip positioned at a 45° angle to create this gradient interface. From then on, the tube must be handled gently so as not to disturb this gradient.
   7. Centrifuge the tube in a prechilled swinging bucket centrifuge at 12,500 g for 20 min with the brake set to OFF.
   8. Just before the centrifugation step is completed, add the RNAse inhibitors to the NSB buffer when proceeding to the scRNA-seq pipelines (see Table 1G).
   9. Following centrifugation, aspirate the supernatant without disturbing the pellet using a vacuum pump.
      NOTE: Using manual pipetting will compromise the quality of the final nuclei suspension.
   10. Using the 1 mL wide-orifice pipette tip, gently resuspend the pellet in 100-300 µL of NSB, and transfer the nuclei suspension into a clean, prechilled 1.5 mL tube.
   11. Count the nuclei using trypan blue solution (1:1 ratio of trypan to sample) and a manual hemocytometer (e.g., mix 10 μL of trypan blue with 10 μL of nuclei suspension, and use 10 μL of the mixture for counting; Figure 2D).
   12. Use the obtained nuclei suspension immediately for the single-nucleus genomics assay.
       ​NOTE: The nuclei suspension in NSB can be stored refrigerated at 4 °C for up to 1 week for further analysis by flow and/or imaging flow cytometry but not for snRNA-seq or snATAC-seq.

3. Nuclei sorting for hepatocyte ploidy profiling or well-based sequencing approaches

1. For flow cytometry-based cell sorting, filter the nuclei suspension through a 50 µm filter into a prechilled 5 mL FACS tube.
2. Use a flow cytometry sorter fitted with a 100 μm nozzle. Load the FACS tube onto the sorter, and preview the sample.
3. Set up the gating strategy for the nuclei sorting, starting with a scatter gate by plotting the forward scatter area versus the side scatter area (FSC-A/SSC-A), followed by Hoechst-Height versus Hoechst-Area (nuclei gate), and then Hoechst-Width versus Hoechst-Area (singlets gate). Visualize the nuclei ploidy profile on the Hoechst-Area histogram.
   NOTE: For better peak resolution, visualize the Hoechst channel (450/50) on the linear scale (Figure 3A).
4. For sorting into 96-/384-well plates, set the droplet delay, and optimize the plate alignment using the colorimetric method with benzidine substrate-horseradish peroxidase (TMB-HRP), as described previously⁶.
5. Set both the sample cooling and plate holder to chill at 4 °C, with the sample rotation turned on at 300 rpm.
6. Sort the single nuclei at a sample concentration of ~1 x 10⁵ nuclei/mL and with the flow rate at 200-500 events/s.

4. Visual inspection and quantification of nuclear parameters by imaging cytometry (optional)

1. Load a 0.5 mL tube containing 50 µL of the nuclei suspension at a concentration of 2 × 10⁷ nuclei/mL.
2. Set up an all events gate based on the aspect ratio versus the Hoechst fluorescence channel to visualize the nuclei ploidy profile (Figure 3B).
3. Acquire the sample at a 40x magnification using brightfield (BF) and the Hoechst fluorescence channel.
4. Inspect and quantify 2n and 4n nuclei using the brightfield measurement and Hoechst fluorescence intensity with imaging cytometry software (Figure 3C).

5. Single-nucleus RNA-seq, ATAC-seq, or multiome library construction and sequencing

1. For droplet-based snRNA-seq approaches, load the purified nuclei suspension directly into the microfluidic device for automated parallel partitioning and molecular barcoding¹⁷.
2. After the microfluidic run is completed in the single-cell partitioning device, collect the gel bead-encapsulated nuclei, incubate, and clean as described previously in the manufacturer's guidelines¹⁷.
3. Perform 11 polymerase chain reaction (PCR) cycles for the cDNA preamplification using the following program: 3 min at 98 °C, (15 s at 98 °C, 20 s at 63 °C, and 1 min at 72 °C) x 11, 1 min at 72 °C, and hold at 4 °C. Continue with an end repair and A-tailing step and adaptor ligation, as indicated by the manufacturer¹⁷. For the subsequent final gene expression library construction, perform 10 PCR cycles using the following program: 45 s at 98 °C, (20 s at 98 °C, 30 s at 54 °C, 20 s at 72 °C) x 10, 1 min at 72 °C, and hold at 4 °C.
4. Sequence the obtained libraries to a read depth of ~20,000-50,000 mean reads per nucleus.
5. For the joint multiomics (RNA + ATAC) droplet-based sequencing, incubate the liver nuclei in lysis buffer for 5 min, and then tagment them for 1 h, as described previously¹⁸.
6. Load the tagmented nuclei directly into a microfluidic device for automated parallel partitioning and molecular barcoding.
7. After the microfluidic run is completed, collect the gel bead-encapsulated nuclei, incubate, and clean as described by the manufacturer¹⁸.
8. Perform six PCR cycles for the cDNA preamplification step using the following program: 5 min at 72 °C, 3 min at 98 °C, (20 s at 98 °C, 30 s at 63 °C, 1 min at 72 °C) x 6, 1 min at 72 °C, and hold at 4 °C.
9. Take 35 µL of the preamplified sample, and perform a cDNA amplification as follows: 3 min at 98 °C, (15 s at 98 °C, 20 s at 63 °C, 1 min at 72 °C) x 6, 1 min at 72 °C, and hold at 4 °C. Continue with the end repair and A-tailing step and adaptor ligation, as indicated by the manufacturer¹⁸ . For the subsequent final sample indexing PCR, perform 15 PCR cycles as follows: 45 s at 98 °C, (20 s at 98 °C, 30 s at 54 °C, 20 s at 72 °C) x 15, 1 min at 72 °C, and hold at 4 °C.
10. For the construction of ATAC libraries, use 40 µL, and amplify for six PCR cycles for sample indexing using the following program: 45 s at 98 °C, (20 s at 98 °C, 30 s at 67 °C, 20 s at 72 °C) x 6, 1 min at 72 °C, and hold at 4 °C.
11. Sequence the obtained multiome gene expression libraries to a minimum read depth of 20,000 read pairs per nucleus and the multiome ATAC libraries to a minimum read depth of 25,000 read pairs per cell, as recommended by the manufacturer¹⁸.