Isolation of Nuclei from Flash-frozen Liver Tissue for Single-cell Multiomics

Here, we present a protocol for isolating nuclei from flash-frozen, archived liver tissues for single-nucleus RNA-seq, ATAC-seq, and joint multiomics (RNA-seq and ATAC-seq).

Transcript

This easy and reproducible protocol was successfully used to isolate high quality nuclei from healthy and diseased mouse model livers, as well as livers from human and non-human primates. This method requires only a small amount of frozen tissue material. Also, density grade incentivization gives a very clean nuclear suspension with all the major cell types in the liver preserved in the final nuclear prep.

This method of nuclei extraction can be used in bio archived human liver samples that are stored in biobanks. Begin the tissue homogenization by cutting a 2230 milligrams or five millimeter cube piece from a liver tissue placed in the Petri dish on dry ice using a pre chilled scalpel. Immediately transfer the Petri dish to wet ice and add one milliliter of homogenization buffer.

Using the cold scalpel, mince the tissue as much as possible, allowing it to aspirate with a one milliliter wide orifice tip. Collect the tissue suspension and transfer it to a pre chilled two milliliter glass dounce Homogenizer. Wash the Petri dish with an additional 0.521 milliliter of homogenization buffer and collect all the remaining tissue pieces while keeping everything on ice.

Slowly and carefully perform five strokes with loose pestle A on ice without creating bubbles. Ensure that the pestle moves carefully from the top to the bottom of the homogenizer with each stroke. Subsequently perform 10 to 15 slow strokes with tight pestle B on ice without creating bubbles.

Once done, filter the homogenate through a 50 micrometer cell strainer while transferring it into a pre chilled 1.5 milliliter tube. Use more than one filter or tube for homogenates containing a high amount of connective tissue clumps. Rinse the homogenizer and the filters with an additional 0.521 milliliter of homogenization buffer to thoroughly collect all the tissue homogenates before the density gradient centrifugation.

For density gradient centrifugation, centrifuge the filtered homogenate in a pre-chilled fixed angle centrifuge at 1000 G for eight minutes at four degrees Celsius. While the sample is getting centrifuged, prepare a 1.5 milliliter tube containing 250 microliters of 50%iodixanol dilution and one two milliliter tube containing 500 microliters of 29%iodixanol dilution. Place both tubes on ice.

Then from the centrifuge suspension, aspirate the supernatant without disturbing the pellet using a vacuum pump. Re-suspend the pellet in 250 microliters of homogenization buffer using the one milliliter wide orifice pipette tip. Then transfer 250 microliters of the nuclei suspension into a pre-chilled 1.5 milliliter tube containing 250 microliters of 50%iodixanol and mix it gently and thoroughly to generate a 25%iodixanol nuclei suspension.

Next, transfer 500 microliters of the 25%iodixanol nuclei suspension into a pre chilled tube milliliter tube containing 500 microliters of 29%iodixanol dilution. Centrifuge the tube in a pre-chilled swinging bucket centrifuge at 12, 500 G for 20 minutes, with the break set to off. Just before the centrifugation step is completed, add the RNAs inhibitors to the nuclei storage buffer, or NSB while proceeding to the single nucleus RNA sequencing pipelines.

After completion of the centrifugation, aspirate the supernatant without disturbing the pellet using a vacuum pump. Gently re-suspend the pellet in 100 to 300 microliters of NSB using the one milliliter wide orifice pippet tip and transfer the nuclei suspension into a clean pre chilled 1.5 milliliter tube. Count the nuclei using trypan blue solution with a manual hemocytometer and immediately use the obtained nuclei suspension for the single nucleus genomics assay.

For flow cytometry based cell sorting, filter the nuclei suspension through a 50 micrometer filter into a pre-chilled five milliliter fluorescence activated cell sorting or fax tube. Use a flow cytometry sorter fitted with a 100 micrometer nozzle and load the fax tube onto the sorter before previewing the sample. 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, followed by hoaxed height versus hoaxed area, and then hoaxed width versus hoaxed area.

Visualize the nuclei ploidy profile on the hoaxed area histogram. For sorting into 96 or 384 well plates, set the droplet delay and optimize the plate alignment using the colormetric method. Set the sample cooling and plate holder to four degrees Celsius with the sample rotation turned on at 300 RPM.

Sort the single nuclei at a sample concentration of approximately one times 10 to the fifth nuclei per milliliter with a flow rate of 200 to 500 events per second. The microscopic examination of the nuclei extracted from frozen livers showed that the density gradient centrifugation step greatly facilitated the removal of unwanted cellular and tissue debris. This methodology preserved all levels of ploidy, which was validated and quantified by cytometric analysis.

High quality metrics were observed from the data obtained using nuclei extraction. Uniform manifold approximation and projection represented the number of counts across all the nuclei and the major cell types, which can confidently be identified only with the nuclear transcriptome and relatively shallow sequencing. This approach permitted the investigation of liver specific transcription factors and downstream target genes involved in the metabolism of xenobiotics.

The complimentary pattern of hallmark genes such as, the percentral CYP2E1, and the periportal CYP2F2 showed that the extracted nuclei retained critical information about liver donation. The gene expression library of RNA was sequenced to a depth of 44, 600 reads per nucleus and the ATAC library to a depth of 43, 500 reads per nucleus. A weighted nearest neighbor analysis performed for multiple measurements of both modalities, RNA plus ATAC, using the Seurat and Signac packages allowed the identification and annotation of the major and minor liver cell types without prominent biases due to cell size or nuclear fragility.

The upstream transcriptional regulators and the hallmark genes of liver donation were also detected in this method. It is important to avoid creating too many bubbles during down's homogenization when moving the pestles up and down, and to stick to the number of strokes indicated in the protocol.

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Journal (Biology)

Journal (Biology)

Isolation of Nuclei from Flash-frozen Liver Tissue for Single-cell Multiomics

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