Single Cell Sequencing Sample Preparation Guidelines

The standard of high quality cell for sequencing

Total number of cells: > 1×10⁵ cells

Small cell number is not enough for accurate quality control, and inaccurate quality control can easily lead to sequencing data quality problems.

Cell concentration: 700-1200 cells/µl

Ensure that a certain volume of high quality cells can be used in the machine, and that the concentration of repeat counts does not fluctuate by more than ±100 cells/µl.

Cell diameter ≤ 5 µm, target cell diameter <40 µm

It is recommended to use cell filters with 40 µm or less pore size to filter out cell debris and clumped cells, while small cell diameters increase the rate of multicellularity and large cell diameters tend to have blocked pores or low capture efficiency.

Cell viability ≥85%

Cell viability is an important reference indicator to reflect RNA integrity, for cells with heavy damage or excessive dissociation does not reflect RNA integrity well, so the higher the cell viability is, in principle, the better.

Suspension condition: use PBS or culture medium without Ca²⁺, Mg²⁺, EDTA to fully resuspend

Wash twice with DPBS and 0.04% BSA. The cell suspension should minimize cell clumping, excessive cell debris, the presence of large debris or the presence of other impurities. No metal Ca²⁺, Mg²⁺, EDTA and other substances affecting the activity of reverse transcriptase are present in the cell-on-board resuspension.

Cell diameter ≤ 5 µm, target cell diameter < 40 µm

It is recommended to use cell filters with pore size of 40 µm and below to filter out cell debris and agglomerated cells. Small cell diameters increase the rate of multicellularity, while large cell diameters tend to have blocked pores or low capture efficiency.

Collection of tissue sample

1. Fresh tissue: sample size ≥ 0.2g
2. Punctured tissue: no less than 2 strips (diameter ≥ 1.5 mm, length 1.5 cm)
3. Very small sample size: please contact technical support, according to the lesion of the sample, sampling method, and sending conditions to give reasonable advice.
4. Notes on tissue sample collection

• Consistent sampling: The collection status of the samples from the experimental and control groups should be the same as possible.
• Accurate sampling: Remove non-target tissues, such as excess fat and envelope, and wash to remove blood residues.
• Keep the sample moist: transfer the sample to tissue preservation solution as soon as possible after sampling. Samples exposed to air for a long time are prone to dehydration and death of cells on the tissue surface. For tissues with small diameters, prolonged exposure may directly result in samples not obtaining sufficient single cell suspension. Examples include puncture samples, biopsy forceps sampling of small diameter granular tissues.
• Avoid defacement: Avoid chemical contamination and thermal damage. If the use of laser knife, electric knife and other sample collection tools is unavoidable, the thermally damaged part should be removed; biopsy sampling is best taken in large pieces each time the sample is clamped to reduce cell damage and irreversible effects on subsequent experiments.

5. Storage and transportation

Fresh tissue samples are required to be fully submerged in pre-cooled tissue preservation solution at 2-8°C.

Cell sample preparation

1. PBMC peripheral blood mononuclear cells

• PBMC cells in blood are usually isolated by Ficoll density gradient centrifugation.
• If single cell sequencing cannot be performed immediately, after extracting PBMC in good condition, you can choose to send them after freezing.

2. Cultivation of cells

• Walled cells are generally dissociated by digestion with 0.25% Trypsin-EDTA enzyme; fragile cell lines are treated with a slightly weaker collagenase to effectively ensure cell viability; suspended cells can be mixed directly by gentle blowing of the cell suspension. Wash the cells by resuspension with PBS containing 5% FBS or 0.05% BSA (or conventional medium).
• Place at 2-8°C and perform single cell sequencing on the machine within 2h. For cells that need to be transported or preserved, they can be lyophilized.

3. Cell freezing

• The standardization of cell freezing has a large impact on the quality of cell freezing, and the gradient freezing procedure needs to be strictly observed.
• 1×10⁶-10⁷ cells are resuspended in 1mL of cell lyophilization solution, transferred into external spiral lyophilization tubes, programmed to cool down to -80°C, and transferred to liquid nitrogen for long-term storage. Frozen cells can be revived for experiments, please consult our technical experts for details.

Organoid sample preparation

Organoids are usually cultured in a matrix gel composed of extracellular matrix, containing some laminin, collagen, growth factors, etc., which can be dissolved into liquid at 4°C and can be re-solidified at 37°C, providing an important microenvironment and tissue support for cell growth in space.

Special sample preparation

• Cell fragile: direct nucleation is recommended to obtain single nucleated suspensions
  For cell fragile tissues, such as liver parenchymal cells or mature adipocytes, which are prone to rupture and have larger cells, it is difficult to get a single cell suspension with a high percentage of target cells by enzymatic digestion, and it is recommended that the tissue be nucleated directly to obtain a single nucleated suspension.
• High fat content: nucleation is recommended
  For tissues with high fat content, the lipid density is low, so it is in floating or suspended state during dissociation and centrifugation, not easy to sink (except immature adipocytes), and mature adipocytes are easily removed after centrifugation and discarding supernatant and collecting through sieve, only some immature adipocytes can sink and enrich during centrifugation and be captured on the machine, but the percentage is limited.
• Cell heterotypes: nucleus aspiration is recommended to obtain single cell nuclear suspensions
  For tissues with cell heterotypes, such as muscle, heart, brain and nerve, the cell diameter is too large to pass through the microfluidic chip, which will easily cause chip pipeline blockage and microdrop generation failure, so it is recommended to choose nucleus extraction to obtain single-cell nuclear sequencing data.
• Easy to degrade: avoid excessive washing and do not transport long distances
  For samples that are easily degraded by themselves, such as pancreas (self-secreted enzyme fluid) and mouse intestine (intestine contains digestive food enzymes), care should be taken to avoid excessive washing and try not to transport them long distance, and digest them on the machine as soon as possible.
• Highly damaged or diseased tissue: contain surrounding tissue for protection
  Both to consider the needs of the subject and to ensure that the tissue is in good condition (e.g., highly damaged or diseased target tissues, sampling often requires more tissue volume, including the surrounding area tissue for protection.
• Non-model animal samples
  In addition to conventional species such as humans, monkeys, mice, rats and other mammals, single-cell sequencing is also widely used in freshwater organisms, marine animals, reptiles, amphibians, avian species, insects, plants, etc. (e.g., zebrafish, jellyfish, snakes, frogs, birds and birds, fruit flies, parasites, hydroids, Arabidopsis, crops, protected plants) Many species need to be sampled, preserved and transported according to the characteristics of the species and their environment. Preservation and transport conditions according to the characteristics of the species and their environment.

Cell sorting

Flow sorting

Flow cell sorting uses fluorescein to label different molecules and distinguishes target cells from non-target cells by adjusting the appropriate voltage, compensation, etc. In the field of single cell sequencing, flow sorting can distinguish specific cell populations and enable quality optimization of single cell suspensions, allowing for more purposeful and accurate studies.

1. Pre-experimentation is recommended before sorting to understand the content percentage of target cells as a way to optimize the sorting conditions of the flow cytometer.
2. Sorting time: depending on the tolerance of the cells to be sorted, to ensure cell viability, it is recommended to finish within one hour.
3. Cell collection solution: 1 x DPBS (or medium), 0.04% BSA / 5% FBS.
4. For flow-sorted cells, the principle of non-essential non-freezing should be followed.

Immunomagnetic bead sorting

Immunomagnetic bead cell sorting is performed by attaching specific antibodies capable of binding to cell surface antigens to magnetic beads. According to the difference of the labeled cells, magnetic bead sorting is usually divided into two categories: positive sorting and negative sorting.

1. For magnetic bead sorting, the distinction and selection of positive and negative sorting kits are particularly important, and attention should be paid to the reliability of sorting reagents and sorting efficiency.
2. pre-experiments to estimate the proportion of target cells to facilitate formal sample preparation.
3. rare cell sorting can be selected using the composite sorting method, which is mainly used for cell subpopulation sorting or high purity cell separation.
4. After sorting, the cells are easily agglomerated, so avoid high-speed centrifugal enrichment.

Sorting considerations

1. Lysis of erythrocytes: if there is red precipitation visible to the naked eye in the original sample, the red cells should be cleaved first, but attention should be paid to the cleavage time to avoid damage to other cells.
2. Count correction: the cell count value of flow sorting may be higher than the real cell total (20-80%), requiring counters or microscopy for count correction.
3. Collecting cells: it is recommended that the centrifuge speed is within 500 g, more than 500 g will easily lead to cell resuspension or clumping. If you are worried about collecting cells, you can increase the centrifugation time.