A protocol for acquiring high-quality single-cell multi-omics data from human peripheral blood

Summary

Single-cell analysis of human peripheral blood cells provides insights into innate and adaptive immune systems. However, robust protocols are essential to ensuring single-cell sequencing data quality and cell viability. Here, we present a protocol for acquiring high-quality single-cell multi-omics data from human peripheral blood mononuclear cells (PBMCs). We describe steps for collecting human blood followed by single-cell sequencing, whole-genome sequencing, and metabolome and proteome analysis of PBMCs using modified multi-omics sample processing.

Graphical abstract

Highlights

• •Steps for obtaining PBMCs with high viability using centrifugation-based isolation
• •Instructions for generating high-quality PBMC suspensions of single-cell sequencing
• •Guidance on constructing a multi-omics library for sequencing and analysis

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Single-cell analysis of human peripheral blood cells provides insights into innate and adaptive immune systems. However, robust protocols are essential to ensuring single-cell sequencing data quality and cell viability. Here, we present a protocol for acquiring high-quality single-cell multi-omics data from human peripheral blood mononuclear cells (PBMCs). We describe steps for collecting human blood followed by single-cell sequencing, whole-genome sequencing, and metabolome and proteome analysis of PBMCs using modified multi-omics sample processing.

Before you begin

This protocol is designed to investigate the cellular heterogeneity as well as gene regulation network of the peripheral blood cells (PBMCs) and to elucidate the mechanisms of immune responses in human cohort. The quality of PBMCs is vital for human cohort study because it is easy to cause cell damage when processing large numbers of samples at the same time. Currently, there are only a few human cohort PBMC studies based on both scRNA-seq and scATAC-seq, which all faces challenges of acquiring high-quality cells efficiently.

Therefore, this protocol describes the specific experimental steps for isolating peripheral blood mononuclear cells (PBMCs) by using the SepMate isolation method, which is a centrifugation-based strategy that allows fast, efficient and reproducible recovery of PBMCs from whole blood with minimal contamination from other blood cell types. In addition, cell fixation was performed immediately after cell recovery to ensure the quality of scATAC-seq data.

In parallel, this protocol incorporates multi-omics sequencing approaches, which is suitable for identifying differential genome profiles, gene expression profiles and chromatin accessibility profiles of peripheral blood cells. With this protocol, we can also detect differential metabolome, lipidome and proteome profiles of plasma. The data obtained via this protocol can be used to get deeper insights of differences in immune function among the human cohort and uncover the key biological processes and mechanisms driving cellular heterogeneity.¹ The protocol provides commercial assays and services for each modality with notes and suggestions as examples but allows for alternative options as long as they meet the same quality and functionality standards. This flexibility allows researchers to tailor the protocol to their specific needs and concentrate on the modalities that are most pertinent to their study.

To conclude, the goal of this protocol is to link the insights of metabolism, immunity, and disease etiology to provide a deeper understanding of the complex interactions within the immune system. It is important to note that special considerations must be taken to ensure the safety of the operators when working with peripheral blood samples for infectious diseases. For example, this protocol has been successfully applied to PBMCs isolated from HIV-infected patients. In that case, the experimenter must wear corresponding personal protective equipments (PPE) to perform sample processing and library construction processes in a biosafety level 2 (BSL2) laboratory.

Institutional permissions

Human peripheral blood samples used for scientific study should be acquired from volunteers or patients with written informed consent and official ethic approval from authorities. The human peripheral blood used in this protocol was collected from BGI Research with informed consent of volunteers and approval from BGI Ethics Committee (Permit No. BGI-IRB 23050-T1).

Prepare before the experiment

Timing: Days to hours before experiment

Prepare all the buffer solutions, mediums, and other necessary materials (Detailed in key resources table or materials and equipment setup).

Prepare on the day of the experiment

Timing: 1 h before experiment

• 1.Put all necessary consumables in the biological safety cabinet. Cleanse and sanitize them by using 75% medical alcohol and DNA-Off, followed by 30 min exposure under UV light sterilization. Upon commencing the experiment, wipe all consumables with RNase-Zap to eradicate RNase contamination.
• 2.Set the temperature of centrifuge to 25°C.
• 3.Turn on the water bath and set the temperature to 37°C.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Chemicals, peptides, and recombinant proteins
1x PBS	Thermo Fisher Scientific	10010023
Fetal bovine serum (FBS)	ExCell Bio	FSP500
Ficoll-Paque PLUS	GE Healthcare	17144003
Red cell lysis buffer	TIANGEN	Y1328
BSA	Sangon Biotech	A600332-0005
Acridine orange/propidium iodide (AO/Pl) solution	Countstar	RE010212
Dimethyl sulfoxide (DMSO)	Sigma	D2650
RPMI 1640	Gibco	C22400500BT
Formaldehyde	Sigma	F8775
Glycine	Sigma	G8898
Sucrose	Sigma	S7903
1 M Tris pH 8.0	Thermo Fisher Scientific	AM9855G
1 M KCl	Thermo Fisher Scientific	AM9640G
1 M MgCl2	Thermo Fisher Scientific	AM9530G
IGEPAL CA-630	Sigma	I8896-50mL
Digitonin	Sigma	D141-100MG
Tween-20	Sigma	85113
Protease inhibitor cocktail	Roche	4693116001
RNase inhibitor	Neoprimaries	LS-EZ-E-00006P
0.1 M DTT	Neoprimaries	LS-EZ-B-00005PB
Transposase	Neoprimaries	LS-EZ-E-00009P
Nuclease-free water	Ambion	AM9932
TE	Ambion	AM9858
DAPI staining solution	Beyotime	C1006
DNA-OFF	Takara Bio	9036
RNaseZap	Ambion	AM9780
Dead cell removal kit	Miltenyi Biotec	130-090-101
DNBelab C series high-throughput single-cell RNA library preparation set V3.0 (TailM 4)	MGI	940-0001818-00
DNBelab C series high-throughput single-cell ATAC library preparation set V1.0	MGI	940-000793-00
MGIEasy magnetic beads blood genomic DNA extraction kit V3.0	MGI	940-000633-00
Other
CellTrics 30 μm, sterile	Sysmex Partec	04-004-2326
Millipore membrane filter, 0.22 μm pore size	Merck	GSWP04700
Transfer pipets, 3 mL	Biologix	30-0138A1
SepMate-50	STEMCELL	86450
Agilent Bioanalyzer 2100	Agilent Technologies	G2939A
Pipette tips, 10 μL	Axygen	T-300-L-R-S
Pipette tips, 200 μL	Axygen	T-200-L-R-S
Pipette tips, 1,000 μL	Axygen	T-1000-L-R-S
Centrifuge tubes, 15 mL	Corning	430791
Centrifuge tubes, 50 mL	Corning	430829
EP tubes, 1.5 mL	VIOX	V1501-C
A2 biological safety cabinet	Esco	AC2-4S1
Cell counter chip	Countstar	CO010101
Cell counter machine	Countstar	Countstar Rigel S3
Centrifugal machine	Eppendorf	5910 Ri
Eppendorf ThermoMixer C	Eppendorf	5382000031
Qubit 4 fluorometer	Thermo Fisher Scientific	Qubit 4.0
Thermal cycler	Bioer	GeneExplorer TC-XP-D
Magnetic separation rack	NEB	S1515S

Note: All the reagents and equipment can be replaced as long as they have same quality and functionality.

Materials and equipment

10% BSA in PBS

Reagent	Final concentration	Amount
BSA	10%	1000 mg
PBS (without Ca2+ and Mg2+)	N/A	10 mL
Total	N/A	10 mL

Note: Filter the solution through a 0.22 μm pore-sized Millipore membrane filter. Make aliquots of 1 mL in 1.5 mL EP tubes. Aliquots can be stored at −20°C for up to 6 months. Avoid repeated freezing and thawing. It can be diluted by PBS to obtain 1% or 0.04% BSA in PBS.

2% FBS in PBS

Reagent	Final concentration	Amount
FBS	2%	20 mL
PBS (without Ca2+ and Mg2+)	N/A	980 mL
Total	N/A	1 L

Note: It can be stored at 4°C for up to 15 days. Inspect it for bacterial contamination by a microscope prior to utilization.

10% FBS in RPMI 1640

Reagent	Final concentration	Amount
FBS	10%	50 mL
RPMI1640	N/A	450 mL
Total	N/A	500 mL

Note: It can be stored at 4°C for up to 15 days. Inspect it for bacterial contamination by a microscope prior to utilization.

100 x Protease inhibitor cocktail

Reagent	Final concentration	Amount
Protease inhibitor cocktail	100 x	1 tablet
Nuclease-free Water	N/A	500 μL
Total	N/A	500 μL

Note: The tablet is hard to dissolve, therefore, vortex it vigorously until it is fully dissolved. It can be stored at −20°C for up to 1 year and don’t leave it at 4°C exceed 30 min.

2% Digitonin

Reagent	Final concentration	Amount
Digitonin	2%	100 mg
DMSO	N/A	5 mL
Total	N/A	5 mL

Note: Aliquots can be stored at −20°C for up to 1 year.

1% Digitonin

Reagent	Final concentration	Amount
2% Digitonin	1%	500 μL
Nuclease-free Water	N/A	500 μL
Total	N/A	1 mL

Note: Make aliquots of 15 μL in 200 μL PCR tubes. Aliquots can be stored at −20°C for up to 1 year. Avoid repeated freezing and thawing.

NIM buffer

Reagent	Final concentration	Amount
1 M Tris pH 8.0	10 mM	500 μL
Sucrose	250 mM	4.3 g
1 M KCl	25 mM	1.24 mL
1 M MgCl2	5 mM	250 μL
Nuclease-free Water	N/A	48.01 mL
Total	N/A	50 mL

Note: Strain the solution through a 0.22 μm pore-sized Millipore membrane filter. It can be stored at 4°C for up to 15 days. Inspect it for bacterial contamination by a microscope prior to utilization.

Homogenization basic buffer

Reagent	Final concentration	Amount
0.1 M DTT	0.1 mM	5.2 μL
100 x Protease inhibitor cocktail	1 x	51.5 μL
RNase inhibitor (40 U/μL)	0.2 U/μL	25.8 μL
10% BSA in PBS	1%	515.5 μL
NIM Buffer	N/A	4.402 mL
Total	N/A	5 mL

Note: Prepare it within half an hour before the experiment.

Note: Two lysis and washing buffers for scATAC-seq are given below. The regular version usually omitting mitochondria genomes (mtDNA) to improve permeability of various cell types and increase the complexity of the library.² When the analysis of mtDNA mutations is required, mtDNA could be preserved. For instance, when analyzing cells from patients with putative clonal malignancies, such as PBMCs from patients with lymphocytic leukemia, the mtDNA mutations can be analyzed to infer clonal relationships between cells.³

10% IGEPAL CA-630/10% Tween-20

Reagent	Final concentration	Amount
IGEPAL CA-630 / Tween-20	10%	1 mL
Nuclease-free Water	N/A	9 mL
Total	N/A	10 mL

Note: Both Tween-20 and IGEPAL CA-630 are required to omit mtDNA, whereas IGEPAL CA-630 alone is sufficient for preserving mtDNA. The surfactant of Tween-20 possesses a high viscosity, so gently pipette 1 mL of reagents by using a sterile and enzyme-free pipette tip. Subsequently, inject the tip directly into 9 mL of Nuclease-free Water, mix it thoroughly and keep it at 25°C until it is fully dissolved, discard the tip and store the reagents at 4°C for up to 6 months.

Homogenization buffer (Omit mtDNA)

Reagent	Final concentration	Amount
Homogenization Basic Buffer	N/A	485 μL
10% IGEPAL CA-630	0.1%	5 μL
1% Digitonin	0.01%	5 μL
10% Tween-20	0.1%	5 μL
Total	N/A	500 μL

Note: Prepare it within half an hour before the experiment.

HB-washing (Omit mtDNA)

Reagent	Final concentration	Amount
Homogenization Basic Buffer	N/A	990 μL
10% Tween-20	0.1%	10 μL
Total	N/A	10 mL

Note: Prepare it within half an hour before the experiment.

Homogenization buffer (Preserve mtDNA)

Reagent	Final concentration	Amount
Homogenization Basic Buffer	N/A	495 μL
10% IGEPAL CA-630	0.1%	5 μL
Total	N/A	500 μL

Note: Prepare it within half an hour before the experiment.

Step-by-step method details

The workflow chart illustrates the entire work flow of multi-omics experiments in this protocol (Figure 1).

Note: The collection, isolation and library construction steps of whole blood sample from infected patients must be carried out in the corresponding biosafety laboratory, and all the operators must wear PPE including gloves, masks, goggles and protective clothing, etc.

Figure 1.

Workflow for the preparation of multi-omics libraries from PBMCs

Human whole blood collection

Timing: within 6 h

Collection and preparation of human whole blood samples.

• 1.
  Blood collection with EDTA anticoagulants.

  • a.
    Collect approximately 10 mL of fresh whole blood, which is anticoagulated with EDTA anticoagulants. Gently shake the tube up and down to mix blood and anticoagulants.

    CRITICAL: The volume of fresh whole blood needs exceed at least 3 mL to ensure obtaining sufficient PBMCs used to perform all multi-omics library construction procedures with this protocol.

  • b.
    Keep it at 25°C until all samples have been collected.
  • c.
    Transfer all samples to the laboratory.

    CRITICAL: It should not exceed 6 h from whole blood collection to blood sample processing. The blood should not be shaken violently to avoid hemolysis.

PBMC isolation and cryopreservation

Timing: ∼2 h

This step ensures that PBMCs are effectively isolated and preserved for use in recovery processes, maintaining their viability and functionality for future multi-omics experiments.

Note: This protocol has been adapted from the SepMate website, previously described in Efthymiou et al.⁴

• 2.
  Plasma separation before PBMC isolation.

  • a.
    Take the 2% FBS in PBS out of the refrigerator and preheat it to 25°C.
  • b.
    Transfer 10 mL of venous blood to a regular 15 mL centrifuge tube.
  • c.
    Centrifuge it at 3000 rpm for 10 min.
  • d.
    Collect the plasma to another 15 mL centrifuge tube, label and store it at −80°C.

    CRITICAL: To prevent aspirating the mononuclear cells, leave 1 mL of plasma on top of the mononuclear cells.

    Note: The collected plasma in step 2-d can be aliquoted into 250 μL/tube to avoid repeated freezing and thawing.

  • e.
    Add 2% FBS in PBS (25°C) into the 15 mL centrifuge tube mentioned in step 2-c. Make up the final volume to 10 mL. Gently mix it by inverting it up and down.

    CRITICAL: When the volume of venous blood in step 2-b is less than 10 mL, add 2% FBS in PBS in step 2-e to make up the final volume to the original volume of the venous blood.

    CRITICAL: Perform subsequent PBMC isolation at 25°C as soon as possible.

• 3.
  PBMC isolation with SepMate tube (Figure 2).

  • a.
    According to the volume of blood being processed, use a 3 mL Transfer Pipets to carefully add density gradient centrifuge solution (Ficoll-Paque) into the 15 or 50 mL SepMate tube through its central hole (Table 1).

    Note: Small air bubbles produced by pipetting in density gradient centrifugation solutions do not affect the effectiveness of the experiment.

  • b.
    Keep the SepMate tube upright and add the diluted sample mentioned in steps 2-e along the wall of the tube using a pipette.

    CRITICAL: The diluted sample could also be poured directly along the wall of the SepMate tube. Be very careful to avoid pouring diluted samples directly into the small center hole.

  • c.
    Centrifuge the mixture at 1200 g for 10 min at 25°C.

    CRITICAL: For samples stored for more than 24 h, the recommended centrifugation time is 20 min.

  • d.
    Quickly pour out the buffy coat into a new 50 mL centrifuge tube, which contains enriched mono-nuclear cells (MNCs).

    CRITICAL: The SepMate tube cannot be inverted more than two seconds.

  • e.
    Add 2% FBS in PBS (25°C) into the new tube mentioned in step 3-d to make up to around 30 mL to wash MNCs.
  • f.
    Centrifuge the tube at 300 g for 8 min at 25°C. Discard the supernatant and be careful to avoid discarding the pellet.
• 4.
  Erythrocyte lysis.

  Note: When the cell pellet is observed to be visibly red, perform the erythrocyte lysis steps.

  • a.
    Add 1 mL of PBS to the tube mentioned in step 3-f and gently resuspend the cell pellet.
  • b.
    Add 3 mL of red cell lysis buffer and thoroughly mix it with cell suspension by gently pipetting.
  • c.
    Incubate the mixture at 25°C for 5 min.
  • d.
    Add 6 mL of PBS and gently shake it to stop the lysis.
  • e.
    Centrifuge the tube at 500 g for 5 min at 25°C.
  • f.
    Carefully remove the supernatant.
• 5.
  Cell counting and viability assay.

  CRITICAL: Microscope is alternative to increase accuracy when counting a small number of samples. However, when counting a large number of samples, the automatic counter is optimal to shorten counting time. Long time operations will reduce the cell viability.

  Note: Counting 2 or 3 samples as representative is recommended with a multitude of samples.

  • a.
    Use PBS to resuspend the cell pellet and stain it by using AO/PI solution.
  • b.
    Transfer 2 μL of cell suspensions, 8 μL of PBS and 10 μL of AO/PI solution into a EP tube, mix the mixture gently by pipetting.
  • c.
    Count cell number and determine cell viability with Countstar Rigel S3.
  • d.
    The quality of PBMCs is determined by the number and viability of cells (Figure 3).

    Note: 3 mL of fresh whole blood normally yield about 3 million PBMCs and the viability is normally over 90%, which are enough for the downstream assays. Unskilled or long-time operation may lead to a lower cell viability. Continuing cryopreserving cells with viability lower than 90% is not recommended.

    Note: Using trypan blue for cell number and viability counting is alternative.

  • e.
    Prepare the cryopreservation solution (10% dimethyl sulfoxide in FBS, about 1.5 mL of cryopreservation solution per sample), store it at 4°C.
  • f.
    Centrifuge PBMCs mentioned in step 5-a at 300 g for 8 min at 25°C.
• 6.
  PBMC cryopreservation (Crucial for acquiring high-viability PBMCs).

  • a.
    Discard the supernatant, resuspend the cell pellets with 1.5 mL of pre-cooled cryopreservation solution, make aliquots of 300 μL in 2 mL cryopreservation tubes and then quickly place them at 4°C.
  • b.
    Store the cryopreserved cells in turn at 4°C for 30 min, −20°C for 30 min, and −80°C for 12 h.

    Note: Directly store it in Mr Frosty or similar freezing container at −80°C can also be used. The cryopreserved cells could be stored at −80°C for short-term storage up to 2 weeks.

  • c.
    Transfer the cryopreserved cells to liquid nitrogen tanks for long-term storage and maintain regular liquid nitrogen replenishment.

    Note: Ensure the cryopreservation tubes remain upright without toppling over during cryopreservation.

Figure 2.

PBMC and plasma isolation procedures

Table 1.

Amount of density gradient centrifuge solution utilized

SepMate tubes	Starting blood sample (mL)	Density gradient centrifuge solution (mL)
15	0.5 - 4.0	4.5
15	> 4 - 5	3.5
50	4 - 17	15

Figure 3.

Cells stained by AO/PI and analyzed with Countstar Rigel S3

Red points indicates dead cells and green points indicates live cells.

PBMCs recovery

Timing: ∼30 min

This step ensures the effective recovery and handling of cryopreserved PBMCs.

• 7.
  Recovery of PBMC samples (Figure 4).

  Note: To avoid contamination of cells, exposure of the skin wrist is not allowed during operation. When the protective gloves touch the area outside the ultra-clean bench, the protective glove surface must be meticulously cleaned with 75% medical alcohol before resuming the experiment.

  • a.
    Take the 10% FBS in RPMI1640 and 0.04% BSA in PBS out of the refrigerator and warm them to 25°C.
  • b.
    Remove the cells from the liquid nitrogen tank and transfer them to the experimental area with liquid nitrogen (This refer to short-distance transferring).

    CRITICAL: Dry ice or liquid nitrogen can be used to transfer PBMCs stored at −80°C. However, when transferring PBMCs stored in the liquid nitrogen tank, the liquid nitrogen used to transfer the samples cannot be replaced by other materials, such as dry ice or shaved ice. The temperature differences will lead to a decrease in sample viability. Therefore, when cells need to be transported between different cities, they are first frozen at −80°C, transported to the destination on dry ice, and then stored in liquid nitrogen for long-term storage.

  • c.
    Add 3 mL of pre-warmed 10% FBS in RPMI1640 to a 15 mL centrifuge tube in advance.
  • d.
    Use tweezers to hold the cryopreservation tube, put it into a 37°C water bath and shake it back and forth for about 60–120 s, quickly melt the ice until tiny ice crystals remains.

    CRITICAL: Use the cryopreservation tube with internal thread. External thread cryogenic vials with plug seal cap or EP tubes are not suggested, otherwise there will be a risk of cryovial exploding in a 37°C water bath.

  • e.
    Transfer the melted cells into the 15 mL centrifuge tube in step 7-c as soon as possible and pipette about 500 μL of medium to wash the cryopreservation tube.
  • f.
    Mix the cells by gently inverting them up and down 5 times, centrifuge the tube at 300 g for 5 min at 25°C.

    CRITICAL: Do not mix the cells by pipetting with tips, which can easily cause cell damages.

  • g.
    Carefully remove the supernatant.

    Note: When the cell pellet is observed to be visibly red, perform the erythrocyte lysis steps as mentioned in step 4-a to 4-f.

  • h.
    Add 500 μL of pre-warmed 0.04% BSA in PBS to resuspend PBMCs.

    Note: When obvious impurities or cell aggregates in the cell suspension were observed, perform the following filtration step, otherwise skip the step.

    Optional: Use 500 μL of pre-warmed 0.04% BSA in PBS to moisten the 30 μm cell strainer, subsequently, filter the cell suspension and collect the filtered cells into a new EP tube.

Figure 4.

Steps of PBMC recovery

Cell counting

Timing: ∼10 min

This protocol provides an approach for accurately assessing cell viability and counting the total number of PBMCs after recovery, which is crucial for ensuring the success of downstream experiments.

• 8.
  Cell counting for library construction.

  • a.
    Follow the step 5b and 5c to perform cell counting.

CRITICAL: The viability should be greater than 80%, and the aggregation ratio should be less than 10%. Otherwise, perform dead cell removal procedures as mentioned in the following step.

Optional: Use Miltenyi Dead Cell Removal Kit to improve cell viability. Find the protocol via miltenyibiotec.com.

Note: The operation of removing dead cells will lose nearly half of the cells so it needs to be chosen when the cell numbers are greater than one million.

scRNA-seq and scATAC-seq library construction

Timing: ∼12 h

This step provides a comprehensive approach to constructing dual-omics libraries for simultaneous single-cell RNA and ATAC sequencing, enabling integrated analysis of gene expression and chromatin accessibility at the single-cell level.

Note: The protocol has been adapted from the Smart-Seq2,⁵ Drop-seq,⁶ Omni-ATAC² and mtscATAC-seq,³ which provides commercial assays from one company as an example. Other companies with similar services are alternative.

CRITICAL: The PBMC fixation step 10-a for scATAC-seq should be performed immediately after cell counting step 8-a to avoid decreasing cell viability of PBMCs. Therefore, at least two operators are required when perform scATAC-seq and scRNA-seq library construction simultaneously.

• 9.
  scRNA-seq library construction.

  • a.
    Transfer part of cells (20,000 cells per library) acquired in step 7-h into a new 1.5 mL EP tube.

    Note: Ensure the transferred cells with high quality (viability > 80%, clumping and impurity rate < 5%).

  • b.
    Immediately follow the instructions of DNBelab C Series High-throughput Single-cell RNA Library Preparation Set V3.0 (TaiM 4) for short-read sequencing library construction. Find the protocol via mgi-tech.com.

    Note: The following experiment steps are recommended to perform in a class 100,000 or 300,000 clean laboratory to avoid cross contamination. The reagent described in following steps can be obtained from the commercial assay as mentioned in step 9-b.

  • c.
    Prepare the cell and bead phase suspensions in a clean bench followed by the manufacture’s protocol.
  • d.
    Add the cell phase suspensions, droplet oil and bead phase suspensions in a scRNA flow cell in sequence.

    CRITICAL: Strictly follow the order as mentioned in step 9-d to add solutions to avoid failure of droplet formation.

  • e.
    Place the flow cell onto a holder inside of the droplet generator to perform droplet generation.
  • f.
    Collect droplets immediately in four tubes of the PCR 8-strip tube to perform droplet-based reverse transcription (RT).

    CRITICAL: One droplet sample was collected in at least four tubes to ensure that the volume of the droplet product in each tube does not exceed 100 μL.

  • g.
    Perform droplets demulsification by breakage reagent and size selection by magnetic beads.
  • h.
    Collect, amplify and purify the cDNA products.
  • i.
    Follow the instructions of DNBSEQ-T7RS High-throughput Sequencing Set (FCL PE100) V3.0 to conduct single-cell RNA sequencing. Find the service and protocol via mgi-tech.com.
  • j.
    Upon obtaining the cDNA, proceed with single-cell full length transcript sequencing to accurately and reliably identify alternative splicing, gene fusion phenomena, and alternative polyadenylation (APA) sites,⁷ etc.
  • k.
    Follow the instructions of Kinnex full-length RNA kit for Kinnex libraries preparation. Find the protocol via pacb.com.

    Note: One commercial assay for single cell sequencing was described above as an example, which can be replaced by other similar products.

  • l.
    Transfer part of cells (1,700 cells per library) acquired in step 7-h into a new 1.5 mL EP tube.
  • m.
    Follow the instructions of Chromium Single Cell V(D)J Reagent Kits for TCR/BCR library construction to detect peripheral TCR and BCR repertoire diversity.⁸ Find the commercial assay and protocol via 10xgenomics.com.

    Note: Other similar commercial assays for TCR/BCR library construction are alternative, such as reagent kits for single cell immune profiling listed in 10xgenomics.com. As long as the cell viability is over 80% in step 7-h, operators can successfully gain high-quality sing-cell data by strictly following the instructions of manufactures.

• 10.
  PBMC fixation for scATAC-seq.

  • a.
    Transfer 300,000 cells acquired in step 7-h into a new 1.5 mL EP tube and supplement PBS to make up to 200 μL. In the event that the cell count is less than 300,000 and more than 50,000, transfer 200 μL of cell suspension instead.

    CRITICAL: When the cell count is less than 50,000, repeat PBMCs recovery steps (step 7-a to 7-h).

  • b.
    Add 0.54 μL of 37% formaldehyde to the cell suspension above, mix it gently by pipetting (no more than 30 s for each sample) and place it at 25°C for 5 min.
  • c.
    Add 1.08 μL of 2.5 M glycine to quench the fixation, mix it gently and incubate it at 25°C for 3 min.
  • d.
    Centrifuge it at 1,000 g for 5 min at 25°C, carefully remove the supernatant.
  • e.
    Add 180 μL of 1% BSA in PBS, mix it gently by pipetting.
  • f.
    Repeat step 10-d and 10-e.
  • g.
    Centrifuge it at 1000 g for 5 min at 25°C.
  • h.
    Set the temperature of centrifuge to 4°C beforehand for the following centrifuge steps as mentioned in cell lysis.
• 11.
  Cell lysis for scATAC-seq.

  Note: Two choices are given for selectively omitting or preserving mitochondria genomes (mtDNA). The mtDNA was usually omitted for regular scATAC-seq or preserved when the analysis of mtDNA mutations is required.

  • a.
    Prepare necessary reagents (including 1% BSA in PBS, homogenization buffer and HB-washing) in advance.
  • b.
    Discard the supernatant of step 10-g, add 100 μL of pre-cooled homogenization buffer (omit or preserve mtDNA), mix it gently by pipetting about 30 times (no more than 30 s for each sample) and incubate it at 4°C or on ice for 3 min.
  • c.
    Add 400 μL of HB-washing for omitting mtDNA or add 400 μL of Homogenization basic buffer for preserving mtDNA, mix it gently by pipetting about 5 times.
  • d.
    Centrifuge it at 1000 g for 5 min at 4°C, discard the supernatant.
  • e.
    Repeat step 11-c and 11-d.
  • f.
    Add 40 μL of 1% BSA in PBS, mix it gently by pipetting and count cells by DAPI staining solution.
  • g.
    Transfer 2 μL of cell suspensions, 8 μL of PBS and 10 μL of DAPI staining solution into a EP tube, mix it gently by pipetting.
  • h.
    Count the cell concentration by CountStar Rigel S3.

    Note: Other automatic counting machines or microscopes are alternative. Microscopes are suitable for small numbers of samples and automatic counting machines are optimal for large number of samples.

• 12.
  scATAC-seq library construction.

  • a.
    Immediately follow the instructions of DNBelab C Series High-throughput Single-cell ATAC Library Preparation Set V1.0 for library construction. Find the protocol via figshare.com.
  • b.
    Transfer 100,000 cells mentioned in step 11-f into a new EP tube to perform Tn5 transposition.

    CRITICAL: When the cell number is smaller than 100,000, transfer all remaining cells for transposition. At least 10,000 cells are required for following droplet generation step. Therefore, when the cell number is smaller than 10,000, repeat the step 10-a to fix other remaining cells.

    Note: Tn5 transposase used in the scATAC-seq protocol can be obtained from Neoprimaries as listed in key resources table. It can also be prepared in-house, following the protocol described by Picelli et al.⁹

  • c.
    Collect transposed cells and prepare the cell and bead phase suspensions by following the instructions described in step 12-a.

    Note: This commercial assay contain all reagents for scATAC-seq library construction.

  • d.
    Perform droplet formation and followed by PCR in droplets.
  • e.
    Perform demulsification, beads selection and library preparation, add scATAC Barcode Primer in each sample to produce DNA library.

    CRITICAL: Record the number of scATAC Barcode Primer, which is vital for differentiating each sample in sequencing and data analyzing.

Multi-omics library construction

Timing: ∼4 h

This section outlines the key steps involved in constructing libraries for whole genome, proteome, metabolome, and lipidome sequencing, each tailored to the specific needs and techniques of the respective omic layer.

• 13.
  Blood genomic DNA extraction for 30 x whole genome sequencing (WGS).

  • a.
    Collect the residual cells (at least 100,000 cells) procured from step 7-h.

    Note: The step is conducted after scATAC-seq and scRNA-seq library construction. When the residual cells are insufficient, repeat PBMCs recovery steps (step 7-a to 7-h).

  • b.
    Centrifuge the cells at 1000 g for 5 min.
  • c.
    Carefully discard the supernatant.
  • d.
    Snap freeze the cell pellets contained in the EP tube in liquid nitrogen, subsequently store them at −80°C up to 3 years.
  • e.
    When all samples have been collected, follow the instructions of MGIEasy Magnetic Beads Blood Genomic DNA Extraction Kit V3.0 for DNA extraction. Find the protocol via mgi-tech.com.

    CRITICAL: The isolated DNA is utilized for 30 x WGS library construction. A quantity of 300 ng DNA suffices for library construction. The DNA concentrations could be measured with Qubit dsDNA Assay Kit by Qubit 4 Fluorometer (Thermo Fisher Scientific).

• 14.
  Plasma extraction for proteome and metabolome analysis.

  CRITICAL: Plasma samples are used for metabolome and proteome analysis. This step can be conducted after other multi-omics experiment mentioned above.

  • a.
    Slowly thaw plasma samples obtained in step 2-d at 4°C.
  • b.
    Transfer 100 μL of thawed samples from step 14-a into a new EP tube and add 400 μL of pre-cooled methanol, mix it by gently inverting it up and down.
  • c.
    Transfer 100 μL of thawed samples from step 14-a into a new EP tube and add 400 μL of pre-cooled isopropyl alcohol, mix it by gently inverting it up and down.
  • d.
    Place the samples obtained in step 14-b or 14-c on ice to react for 2 h.
  • e.
    Collect the inactivated samples in step 14-b and 14-c.
  • f.
    Centrifuge the samples at 20,000 rpm for 20 min at 4°C.
  • g.
    For samples obtained from step 14-b, collect the supernatant (metabolome samples) and the precipitant (proteome samples).

    Note: The samples of metabolome can be detected by HM700 based on LC-MS/MS, Find the sequencing service via bgi.com. The samples of proteome can be detected by Olink based on proximity extension assay. Find the sequencing service via bgitechsolutions.com. The metabolome and proteome correlation analysis can be conducted by BGI multi-omics services, Find the service via bgi.com.

    Note: This protocol provides one feasible sequencing company as an example. Other companies with similar services are alternative.

  • h.
    For samples obtained from step 14-c, collect the supernatant (lipidome samples) and the precipitant (proteome samples).

    Note: The samples of lipidome can be detected by HML1600 based on LC-MS/MS, Find the sequencing service via bgi.com.

  • i.
    When all samples have been collected, send samples to BGI Tech to acquire multi-omics data.

    Note: The metabolomics data could be analyzed by a flexible and comprehensive software named metaX.¹⁰

Expected outcomes

The comprehensive sample processing protocol is optimized to obtain multi-omics data of human peripheral blood. Cells with a cell viability more than 80% can be obtained in this protocol, which is vital for producing high-quality data of single-cell sequencing (Figures 5 and 6). The experimenters can successfully gain high-quality sing-cell data by strictly following the instructions of manufactures as long as the cell viability is over 80%. Therefore, the protocol provides one commercial assay for each service as an example. Other similar products or services are alternative. It would not be recommended to sequence when the yield of DNA library is lower than 200 ng.

Figure 5.

Examples of PBMC library size profiles from scRNA-seq

Figure 6.

Examples of PBMC library size profiles from scATAC-seq

The example of poor outcome is shown in following problem 6 and problem 7 (troubleshooting). In addition, the quality control parameters of sequencing data are provided in Table 2 and Table 3.

Note: The poor outcomes of scRNA-seq showed low cell number and fraction of mapped reads, which mainly caused by low cell viability (< 80.0%). The potential solution of this is described in problem 2 as well as problem 6 (troubleshooting).

Note: The poor outcomes of scATAC-seq showed low fragment fraction overlapping peaks (FRIP), fragment fraction overlapping TSSs and peak numbers. The most likely reason of this is the invalid fixation. The potential solution of this is described in problem 7 (troubleshooting).

Table 2.

Quality control of scRNA-seq

Sample	Cell number	Median UMI counts/cell	Median genes/cell	Mean reads/cell	Mapped reads	Exonic regions mapped reads
Expected outcomes
Example 1	8,645	4,506	1,736	91,525	93.11%	80.80%
Example 2	8,820	4,122	1,582	101,837	92.47%	78.70%
Example 3	11,136	4,339	1,572	68,168	91.32%	77.40%
Poor outcomes
Example 4	3,490	2,122	867	27,277	40.57%	73.10%
Example 5	2,591	2,318	944	23,474	34.48%	73.50%
Example 6	3,499	2,431	945	36,695	38.79%	80.70%
Standard	> 6,000	> 2,500	> 1,000	> 30,000	> 80.0%	> 60.0%

Table 3.

Quality control of scATAC-seq

Sample	Cell number	Median fragments/cell	Median fragment fraction overlapping peaks	Median fragment fraction overlapping TSSs	Mapped reads	Called peak number
Expected outcomes
Example 1	6,789	12,641	83.41%	50.13%	92.58%	81,665
Example 2	6,435	10,740	84.93%	55.79%	91.68%	81,160
Example 3	7,153	9,844	85.99%	54.84%	93.23%	80,919
Poor outcomes
Example 4	4,052	7,489	19.55%	15.18%	94.55%	41,377
Example 5	5,170	10,682	7.79%	7.40%	94.72%	40,025
Example 6	7,428	7,058.5	16.44%	11.19%	94.64%	46,349
Standard	> 3,000	> 4,000	> 70.0%	> 30.0%	> 80.0%	> 60,000

The commercial assays listed in this protocol can be substituted with other multi-omics kits that meet similar quality and functionality standards. Data from these various multi-omics platforms, when applied to the same sample, can be integrated and analyzed using widely-used software tools. For instance, upstream analysis can be conducted with CellRanger or DNBC4Tools, while downstream analysis can be performed using Seurat¹¹ and Signac.¹²

Limitations

Owing to the varied origins of freshly isolated PBMCs, the isolation and recovery procedures may alter the cells’ thermodynamic, chemical and physical milieu, potentially compromising their viability. Simultaneously, the quality of single-cell suspension derived from PBMCs is crucial for scATAC-seq. Thus, it is imperative to promptly execute the fixation of PBMCs immediately after the recovery steps. Besides, operators are recommended to allocate ample time to ensure the sample processing is conducted seamlessly. Furthermore, the precision and technical proficiency of the operators are paramount in the acquisition of high-quality scATAC-seq data.

Troubleshooting

Problem 1

The cells in suspensions may aggregate during experiments.

Potential solution

The cell suspensions need to be filtered through a 30 μm cell strainer while observing aggregated cells or impurities. The cell strainer can be placed on the centrifuge tube (shown in Figure 7).

Figure 7.

Schematic diagram of filtering cells

Problem 2

The viability of PBMCs is low after thawing.

Potential solution

Ensure the cryopreservation of cells is performed through slow gradient cooling steps for a long period and the recovery process is performed instantly in a short period. In addition, the PBMC isolation steps should be completed within 6 h after sample collection. Thirdly, the time of cell counting cannot be too long.

Problem 3

The nuclear condensed after lysis step 11-f.

Potential solution

Prepare the homogenization buffer on the day of the experiment and strictly control the pipette times and lysis time to effectively lyse cells. All the operations of mixing cell solutions should be handled gently.

Problem 4

Low cell yields after centrifugation steps.

Potential solution

The centrifugal force and centrifugation time can be appropriately and accordingly increased when a small amount of cell pellets is observed after centrifugation. Additionally, the steps of discarding the supernatant should be performed patiently and carefully to avoid aspirating cells.

Problem 5

A low number of cells identified via scRNA/scATAC sequencing.

Potential solution

The most likely reason of this is inaccurate cell counting, resulting in an insufficient number of cells being loaded onto the microfluidic chips. To address this problem, gently pipette the cell suspensions to achieve the greatest possible homogeneity prior to cell counting. When the quantity of samples to be processed is minimal, cell counting may be conducted twice to ensure accuracy.

Problem 6

PBMC library size profiles of scRNA-seq indicated enrichment of short fragments (400–600 bp, etc.) (shown in Figure 8).

Figure 8.

Example of poor outcome from scRNA-seq

Potential solution

The most likely reason of this is the sample processing does not meet the standards, for instance, the cell viability is lower than 80% after cryopreservation or recovery. However, the cells continue to be used for single-cell sequencing library construction, causing the oligo on the magnetic beads to capture large amount of short RNA. At the same time, adapters were added in the subsequent library construction, which may ultimately lead to a low comparison rate. To address this problem, pay attention to the temperature of transportation and centrifugation during the cryopreservation or recovery process. Do not make the temperature differences too large to damage the cell viability.

Problem 7

PBMC library size profiles of scATAC-seq showed unclear periodicity of nucleosome (350–700 bp, etc.) (shown in Figure 9).

Figure 9.

Example of poor outcome from scATAC-seq

Potential solution

The most likely reason of this is the fixation is invalid or the lysis time is too long. The formaldehyde used for fixing PBMCs must be preserved well at 25°C away from light. Besides, the amount of components in lysis and washing buffer should be added correctly, and the lysis time cannot exceed 3 min.

Resource availability

Lead contact

Further information and requests for resources and reagents could be directed to and will be fulfilled by the lead contact, Dr. Chuanyu Liu (liuchuanyu@genomics.cn).

Technical contact

Technical questions about this protocol could be directed to the technical contact, Dr. Yue Yuan (yuanyue@genomics.cn) and Dr. Jianhua Yin (yinjianhua@genomics.cn).

Materials availability

This study did not generate new unique materials or reagents.

Data and code availability

This study did not generate any unique code or datasets.

Acknowledgments

This research was supported by the China Postdoctoral Science Foundation (no. 2023M732369). The sequencing services for this study were provided by China National GeneBank, and the commercial assays were produced by MGI. The cartoon picture materials in the experiment process diagram were purchased from BioRender.com.

Author contributions

S.D., G.M., and Z.Y. finished conceptualization, methodology, writing, and editing; S.D., J.C., and X.S. conducted the experiment(s) with assistance from W.Z., Q.D., and Y.W.; J.Y., Y.Y., and C.L. provided project administration and supervision. All the authors reviewed and approved the final manuscript.

Declaration of interests

The authors declare no competing interests.