Optimized Nuclei Isolation from Fresh and Frozen Solid Tumor Specimens for Multiome Sequencing

Summary

The protocol provides a reliable and optimized approach to the isolation of nuclei from solid tumor specimens for multiome sequencing using the 10x Genomics platform, including recommendations for tissue dissociation conditions, cryopreservation of single-cell suspensions, and assessment of isolated nuclei.

Abstract

Multiome sequencing, which provides same-cell/paired single-cell RNA- and the assay for transposase-accessible chromatin with sequencing (ATAC-sequencing) data, represents a breakthrough in our ability to discern tumor cell heterogeneity-a primary focus of translational cancer research at this time. However, the quality of sequencing data acquired using this advanced modality is highly dependent on the quality of the input material.

Digestion conditions need to be optimized to maximize cell yield without sacrificing quality. This is particularly challenging in the context of solid tumors with dense desmoplastic matrices that must be gently broken down for cell release. Freshly isolated cells from solid tumor tissue are more fragile than those isolated from cell lines. Additionally, as the cell types isolated are heterogeneous, conditions should be selected to support the total cell population.

Finally, nuclear isolation conditions must be optimized based on these qualities in terms of lysis times and reagent types/ratios. In this article, we describe our experience with nuclear isolation for the 10x Genomics multiome sequencing platform from solid tumor specimens. We provide recommendations for tissue digestion, storage of single-cell suspensions (if desired), and nuclear isolation and assessment.

Introduction

As our knowledge of tumor biology grows, the importance of analyzing heterogeneous cells across the tumor microenvironment has also increased^1,2. The ability to acquire single-cell RNA and the assay for transposase-accessible chromatin with sequencing (ATAC-sequencing) data from the same cell in a paired-cell fashion (multiome sequencing) provides a significant advance towards this end^3,4. These experiments are expensive and time-consuming, however, and the quality and impact of the data acquired are highly dependent on the quality of the experimental conditions and materials. Standardized protocols for nuclei isolation have been published^5,6. Fresh and heterogeneous tissues require protocol optimization since freshly isolated cells from solid tumor specimens are more fragile than those isolated from cell lines.

Another consideration is that for solid tumors, surgical specimens are often not available from the operating room until late in the day. As such, it is generally not feasible to proceed directly from sample acquisition to nuclei capture without a cryopreservation step. In our experience, freezing a single-cell suspension yields the highest-quality nuclei (rather than flash-frozen whole tissue or other modalities of preservation). This is particularly true for enzymatic tissue types with high RNase content such as the pancreas.

Tissue digestion conditions also need to be designed to maximize cell yield without sacrificing quality⁷. In the context of solid tumor types with dense desmoplastic matrices⁸, the extracellular matrix must be gently broken down for cell release. Additionally, because the cell types isolated are heterogeneous, conditions should be adjusted to support the total cell population. Human pancreatic cancer (pancreatic ductal adenocarcinoma) samples are used in the described protocol. Pancreatic cancer represents a highly desmoplastic tumor type, which portends relatively sticky tissue and cells. Moreover, as pancreatic tumor specimens available for research also tend to be relatively small, efforts are made to maximize the quantity of cells captured.

Isolation of nuclei requires the most optimization in terms of cell lysis conditions and timing, as well as reagent types and ratios. Handling the nuclei over the course of isolation also requires great care. In this article, we describe our experience optimizing nuclear isolation for the 10x Genomics multiome sequencing platform from solid tumor tissue (Figure 1). We provide recommendations for tissue digestion, cryopreservation of single-cell suspensions (if desired), and nuclear isolation.

Protocol

Human pancreatic cancer (pancreatic ductal adenocarcinoma) samples were acquired according to an IRB-approved protocol in our laboratory. Informed consent was obtained from patients for tissue collection. Tissue was transported from the operating room to the laboratory and then processed as follows.

1. Tissue dissociation (digestion)

1. Prepare Digest Buffer (see Table of Materials).
2. Obtain the tissue of interest as soon as possible after tumor excision and transport the specimen in quench buffer (DMEM F-12 with ~5% fetal bovine serum) or 1x PBS on ice.
3. Mince the tissue using clean forceps and scissors in 3-5 mL of Digest Buffer in a 90 mm Petri dish (Figure 2A).
4. Transfer the minced tissue to a 50 mL conical tube and dissociate in ~10 mL of Digest Buffer for 30 min at 37 °C using a water bath with shaking function or a dry agitator.
5. Remove from the agitator and quench with ~20 mL of quench medium. Filter the solution through a 100 µm cell strainer into a clean conical tube.
6. Collect any remaining solid tissue from the strainer (re-mince remaining tissue pieces if needed), and place in ~10 mL of fresh Digest Buffer for another 30 min at 37 °C with agitation. Repeat until all tumor tissue has been dissociated.
7. Pool cell suspension aliquots and filter through a 70 µm followed by a 40 µm cell strainer into a clean conical tube on ice.
8. Centrifuge to pellet cells at 500 × g for 5 min at 4 °C and then decant the supernatant.

2. Cryopreservation

1. Rinse the cells by resuspending the pellet in 1 mL of 1x PBS.
2. Transfer the cell suspension (~1-10 million cells/tube for optimal freezing) to a 1.5 mL cryovial on ice.
3. Centrifuge to pellet cells at 500 × g for 5 min at 4 °C and then decant the supernatant.
4. Resuspend the cells in 1 mL of Bambanker solution, transfer to a 1.5 or 2 mL cryovial (Figure 2B), and freeze using a -1 °C/min cooling rate freezing container (containing 100% isopropyl alcohol) at -80 °C, or transfer to liquid nitrogen if longer-term storage of the sample is anticipated.

3. Nuclei isolation

1. Rapidly thaw the cryovial of cell suspension and place it on wet ice.
2. Transfer the thawed cell suspension to a 2 mL microcentrifuge tube and top up the vial to 2 mL solution with 1x PBS to rinse the cells.
3. Obtain a manual cell count using a hemocytometer to assess both the quantity and quality of the cells (Figure 2C).
4. Centrifuge to pellet cells at 500 × g for 5 min at 4 °C and then gently decant the supernatant using progressively smaller pipette tips to leave behind a dry pellet and maintain it on ice.
   1. Use a swing-bucket rotor for all centrifugation steps. For maximum cell yield, place the tubes in the centrifuge with the hinge facing outwards and then decant with the pipette tip directed towards the opposite side of the tube (Figure 2D).
      NOTE: For decanting the supernatant, use progressively smaller pipette tips to remove fluid to limit disruption of the pellet while maximizing the volume of fluid decanted. This strategy is particularly helpful later in the protocol following nuclei extraction since the nuclei pellet is generally not visible.
5. Prepare 1x Cell Lysis Buffer, Cell Lysis Dilution Buffer, and Wash Buffer (Figure 3A) according to the published protocol with the following modifications (see Table 1, Table of Materials):
   NOTE: Place the Digitonin on a heating block at 65 °C prior to use to dissolve the precipitate (Figure 3B). This typically takes about 10 min.
6. Prepare the 0.1x Cell Lysis Buffer by combining 100 µL of 1x Cell Lysis Buffer and 900 µL of Cell Lysis Dilution Buffer, pipette gently to mix, and place it on ice.
7. Resuspend the cell pellet in 100 µL of chilled 0.1x Cell Lysis Buffer and gently pipette to mix 5x.
8. Incubate on ice for 3 min (Figure 3C).
9. Add 1 mL of chilled Wash Buffer and pipette up and down to gently mix 5x.
10. Centrifuge to pellet the nuclei at 500 × g for 5 min at 4 °C, gently decant the supernatant to a dry pellet, and maintain on ice.
    NOTE: If the starting cell number is low (100,000-200,000 cells), perform the cell lysis and wash steps in a 200 µL PCR tube instead of a 2 mL microcentrifuge tube to decrease the tube surface area and minimize losses. In this case, adjust the Wash Buffer volume down to accommodate the smaller tube volume.
11. Repeat Steps 3.9-3.10 2x for a total of three washes.
12. Manually count the nuclei using a hemocytometer slide under the microscope. Use trypan blue dye if desired to provide contrast for viewing the nuclei and to help discern nuclei from unlysed cells.
13. Prepare the 1x Nuclei Buffer per the published protocol: 20x Nuclei Buffer stock, 1 mM DTT, 1 U/µL RNase Inhibitor in nuclease-free water and keep on ice (see Table of Materials).
14. Resuspend the nuclei pellet in 1x Nuclei Buffer based on goal-targeted nuclei recovery.
    NOTE: If the concentration is high enough, aim for a targeted recovery of 10,000 or slightly higher at this step (3,230-8,060 nuclei/µL) when determining the starting resuspension volume, given the anticipated volume loss of 25 µL and the 20% decrease in concentration with filtering (Step 3.15).
15. Gently pass the resuspended nuclei volume through a 40 µm Pipette Tip Cell Strainer and place the nuclei on ice (Figure 3D).
16. Recount the nuclei (Figure 4A-C). Dilute the final solution further with Nuclei Buffer if needed.
17. Transport the nuclei on ice and immediately proceed with nuclei capture per published protocols.

Representative Results

To isolate high-quality nuclei from patient solid tumor specimens for multiome sequencing (Figure 1), the tumor tissue was dissociated and a single-cell suspension was cryopreserved (Figure 2A-D). The cell suspension was then thawed at the time of planned multiome capture. Nuclei capture was conducted with optimized lysis buffer reagents and timing to maximize both quality and yield (Figure 3A-D). Representative nuclei images show appropriate size and shape (Figure 4A-C). The nuclei circled in pale grey show mild stippling of the envelope.

Figure 1: Schematic of nuclei isolation workflow. Schematic showing the basic workflow from a whole tumor tissue specimen to a single-nucleus suspension ready for submission for nuclei capture, multiome library preparation, and ultimately scRNA- and ATAC-sequencing. Abbreviations: scRNA = single-cell RNA; ATAC-seq = assay for transposase-accessible chromatin with sequencing. Please click here to view a larger version of this figure.

Figure 2: Tissue digestion, cryopreservation, cell assessment, and centrifugation steps. (A) Equipment setup for mincing tumor tissue specimens; (B) cell suspension cryopreservation; (C) microscopic evaluation of cell suspension; (D) approach to centrifuging for this protocol. Please click here to view a larger version of this figure.

Figure 3: Preparation of reagents, nuclei extraction, and filtering. (A) Preparation of nuclei extraction reagents; (B) dissolution of digitonin at 65 °C prior to use; (C) incubation of samples for cell lysis on ice; (D) filtering of extracted nuclei. Please click here to view a larger version of this figure.

Figure 4: Representative microscopic images of nuclei harvested from complex tumor tissue specimens. (A-C) Representative images of nuclei harvested with and without Trypan Blue staining. (B,C) The nuclei circled in pale grey show mild stippling of the nuclear envelope. 10x and 40x objectives used. Scale bars = 50 µm. Please click here to view a larger version of this figure.

Solution Name	Components
Digest Buffer	0.5-1 mg/mL collagenase type IV
	100 U/mL DNase I
	0.1% Poloxamer 188
	20 mM HEPES
	1 mM CaCl2
	3-5% fetal bovine serum (FBS) in Medium 199
1X Cell Lysis Buffer	10 mM Tris-HCl (pH 7.4)
	10 mM NaCl
	3 mM MgCl2
	2% BSA (rather than 1%)
	0.10% Tween-20
	0.1% Nonident P40 Substitute
	0.01% Digitonin
	1 mM DTT
	1 U/µL RNAse inhibitor in Nuclease-free water
Cell Lysis Dilution Buffer	10 mM Tris-HCl (pH 7.4)
	10 mM NaCl
	3 mM MgCl2
	2% BSA
	1 mM DTT
	1 U/µL RNAse inhibitor in Nuclease-free water
Wash Buffer	10 mM Tris-HCl (pH 7.4)
	10 mM NaCl
	3 mM MgCl2
	2% BSA
	0.10% Tween-20
	1 mM DTT
	1 U/µL RNAse inhibitor in Nuclease-free water

Table 1: Solutions used in this protocol.

Discussion

Untangling the heterogeneous cell populations present in the tumor microenvironment is an active area of focus in cancer biology. Similarly, complex tissues exist in benign pathologies such as wound healing and fibrosis. Multiome sequencing has emerged as a powerful tool permitting the acquisition of same-cell paired scRNA- and ATAC-seq data. This protocol describes the isolation of nuclei, which demands optimization in the setting of processing fresh, fragile, small tumor specimens. Here we provide a protocol for nuclei isolation from desmoplastic solid tumor tissue specimens. We have found this approach to yield reliable and high-quality data even when cell suspensions are frozen prior to nuclei isolation and capture.

Increasing the albumin concentration throughout the assay is helpful in limiting cell and downstream nuclei clumping, which can be a problem when working with tumor samples. In terms of tissue digestion, digestion kit reagents can also be used with this protocol if desired (see Table of Materials). If used, we would still recommend following the every-30-min digest/quench and buffer exchanges to maximize the yield of viable cells. Following tissue dissociation, red blood cell lysis can be performed per the manufacturer's protocol. A gradient cell separation protocol can also be performed at the discretion of the researcher (see Table of Materials). We do not perform these protocols routinely, but we have applied such protocols to similar samples without notable differences in results. If significant, small debris is noted after nuclei filtering, Fluorescence-Activated Nuclei Sorting (FANS) can be considered for further purification. A detailed discussion of FANS, including protocol recommendations, is available but beyond the scope of this protocol. Of note, there are protocols in the literature that consider a fixation step prior to cell lysis. However, we prefer to proceed with non-fixed tissue and nuclei as scATAC-seq works best on unfixed tissue⁹.

For cell lysis, as the RNase inhibitor is quite costly, it is reasonable to scale down the prepared volume of the Cell Lysis Buffer (and the Cell Lysis Dilution Buffer) compared with the volumes provided in the published protocol, if desired, as long as the ratios of reagents stay proportional. When assessing and counting the harvested nuclei, a LIVE/DEAD Viability/Cytotoxicity Kit can be applied at the discretion of the researcher. Trypan blue dye can also be used to provide contrast for viewing the nuclei and to help discern nuclei from unlysed cells if needed. Finally, it is worth noting that we have applied the same cell lysis conditions with adjusted reagents per protocol for single-cell ATAC-seq with excellent results¹⁰.

Critical steps in the protocol are precise timing of lysis and gentle but expedient handling of the nuclei. Undermixing will lead to incomplete or inconsistent cell lysis; however, overmixing will shear the chromatin. As noted in other nuclei isolation protocols, nuclei mixing should be accomplished by pipetting rather than vortexing of the sample as the shearing forces can damage the fragile nuclei¹¹. In this regard, it is important that the Cell Lysis Buffer be prepared fresh just prior to use on each occasion. Pipette Tip cell straining (filtering) after the final centrifugation step is key in our experience to prevent nuclei clumping prior to capture. This filtering can be repeated prior to loading if clumps are still detected on the final count or develop during transport of the specimen prior to loading, keeping in mind the additional loss in volume and concentration with each filtering step added. Of note, there should be no significant time pause between nuclei isolation and capture.

Limitations of this protocol are that tissue dissociation and particularly, the cell lysis incubation timing and conditions may need to be optimized for different solid tumor types¹². This protocol has been optimized for desmoplastic solid tumors such as breast carcinoma and pancreatic adenocarcinoma and has also been successfully applied to non-tumor desmoplastic tissues such as parenchymal fibrosis, but other tumor types may require additional adjustments. It is optimized for both fresh cell suspensions as well as frozen cell suspensions with excellent nuclei yield from both of these. We recommend pursuing such optimization sequentially starting with tissue dissociation, and once an optimal single-cell suspension has been achieved, moving on to the optimization of the cell lysis conditions to yield an optimal single-nucleus suspension.

Single-cell ATAC-sequencing and more recently, multiome sequencing present a tremendous breakthrough tool for untangling solid tumor cellular heterogeneity. Cell subtypes can be delineated based on both their chromatin accessibility and gene expression characteristics, and the influence of transcription factor accessibility and expression on tumor behavior can be examined directly. These methodological advances are being broadly applied given their tremendous potential to uncover novel therapeutic targets. As such, the development of optimized protocols to obtain these data is of utmost importance. Here we share this protocol for nuclei isolation in the context of pancreatic cancer tissue-a cancer type for which few therapies exist and all with limited efficacy to date.

Materials

Name	Company	Catalog Number	Comments
100, 70, and 40 μm Falcon cell strainers	ThermoFisher
10x Genomics Nuclei Buffer (20x)	10x Genomics	2000153/2000207
Bambanker	Wako, Fisher Scientitic	NC9582225
BSA	Miltenyi Biotec	130-091-376
Calcium Chloride	Sigma Aldrich	499609
Collagenase (Collagenase Type IV)	ThermoFisher	17104019
Digitonin	Thermo Fisher	BN2006
DNase I	Worthington	LS006330
DTT	Sigma Aldrich	646563
Dulbecco’s Modified Eagle Medium F-12	Thermo Fisher	11320082
Fetal Bovine Serum	Thermo Fisher	10438026
Flowmi 40 μm  Pipette Tip Cell Strainer	Sigma Aldrich	BAH136800040
HEPES	Sigma Aldrich	H3375
Histopaque-1119 Gradient Cell Separation solution	Sigma Aldrich	11191
Medium 199	Sigma Aldrich	M2520
MgCl2	Sigma Aldrich	M1028
Miltenyi GentleMACSTM digest kit
NaCl	Sigma Aldrich	59222C
Nalgene Cryo "Mr. Frosty" Freezing Container	ThermoFisher	5100-0001
Nonident P40 Substitute	Sigma Aldrich	74385
Poloxamer 188	Sigma	P5556
Rnase inhibitor	Sigma Aldrich	3335399001
Tris-HCl	Sigma Aldrich	T2194
Tween-20	Thermo Fisher	85113