Single-Cell Sequencing & Spatial Transcriptomics Sample Submission Guidelines

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How to Get Started

• Consultation: Contact our team for a personalized consultation to discuss your project goals, sample types, and any specific requirements.
• Sample Submission: Follow our detailed Sample Submission Guidelines to ensure the proper collection, preservation, and transportation of your precious samples.
• Project Execution: Once samples are received, our team will execute the project following established protocols, maintaining the highest quality standards.
• Data Delivery: Receive comprehensive data sets, accompanied by detailed analyses, enabling you to derive meaningful insights from your research.

The success of single-cell sequencing and spatial transcriptomics projects heavily relies on the careful collection, preservation, and transportation of precious biological samples. This guide provides step-by-step instructions to ensure the integrity of your samples throughout the entire process. For 10X genomics single cell sequencing, please read this Guide 10x Single Cell Suspension Preparation for Sample Preparation.

Given the diversity of sample types, it is recommended to consult our technical team before submitting.

SAMPLE SUBMISSION & PREPARATION GUIDELINES

Single-Cell Fresh Tissue Samples

The cornerstone of successful cell labeling lies in the utilization of high-quality cell suspensions. As such, the primary objective in single-cell sequencing is to procure fresh tissue samples and meticulously maintain them until the suspension preparation stage.

• Sample Preparation Guide

For effective preservation, employ a tissue preservation solution and store the samples at 4°C. Before usage, verify that the solution remains clear and colorless, devoid of any precipitation.

Surgical or laboratory procedures involve isolating the target tissue, eliminating unrelated surrounding tissues, and sectioning the tissue into small 0.5 cm × 0.5 cm pieces using a sterile scalpel. Typically, these pieces resemble the size of a soybean, or for mouse organs, they can be kept intact. Collect 4-5 such tissue pieces, totaling approximately 200-400 mg in weight.

Subsequently, wash the collected tissue pieces in PBS or saline, and swiftly transfer them into pre-cooled Tissue Preservation Liquid at 4°C. Ensure complete submersion of the tissue in the preservation solution. Fill the preservation tube with the solution, preventing the formation of air bubbles, and seal the cap tightly with a sealing film.

For puncture samples, obtain 3-4 strips measuring 1 mm or more in diameter and 1 cm or more in length without cutting them. Swiftly immerse these strips into the 4°C pre-cooled tissue preservation solution, following the same procedure as with fresh tissue.

• Shipping

To uphold the quality of subsequent single-cell suspension, promptly send the samples for processing (within 48 hours). Opt for ice-crushing transportation or use ice bags during shipping to maintain the desired temperature.

Single-Cell Frozen Samples

For various intricate reasons, such as constraints imposed by experimental design, the structural complexity of sample tissues, or the need to safeguard precious clinical samples that cannot be promptly processed, the option of utilizing frozen tissues for single-cell nuclear transcriptome projects becomes imperative. Typically, these frozen samples are rapidly frozen in liquid nitrogen and transported on dry ice. Below is an illustrative procedure using a mouse lung sample to outline the preparation of routine frozen samples.

• Sample Preparation Guidelines

Employing the liquid nitrogen quick-freezing technique, samples are stored at -80°C for long-term preservation.

Under sterile conditions, commence by isolating the target tissue and eliminating any surrounding tissues unrelated to the experimental samples. Utilize a sterile scalpel to cut the tissues into pieces measuring 0.5 cm × 0.5 cm (approximately the size of a soybean). Collect 4-5 small tissue pieces, totaling about 200 - 400 mg. Subsequently, wash the tissues in PBS or saline, remove excess liquid from the tissue surface, and promptly transfer them into pre-cooled cryopreservation tubes. Conduct an immediate liquid nitrogen flash-freezing for a duration exceeding 15 minutes, followed by long-term storage at -80°C. It is crucial to execute these steps swiftly to preserve sample integrity.

Before further experiments, the frozen tissues must undergo RNA extraction and quality control, ensuring that subsequent procedures commence only after the RNA Integrity Number (RIN) value meets the required standards.

• Shipping

To ensure the integrity of the samples during transit, employ dry ice for delivery. Tailor the quantity of dry ice based on the specific regions involved. This precautionary measure prevents the risk of insufficient dry ice delivery due to regional or weather-related factors, which could potentially compromise the results of subsequent experiments.

Single-Cell Liquid Samples

• Sample Preparation Guidelines

Place the collected single-cell liquid samples in an EDTA anticoagulation tube and store them at 4°C.

Ensure a 4-5 ml blood collection using an EDTA anticoagulation blood collection tube with a purple cap. After sampling, invert the tube 3-5 times to guarantee proper mixing of the EDTA anticoagulant. Immediately after sampling, place the tube in 4°C preservation.

• Shipping

Maintain a transportation temperature of 2-8°C. If using an ice pack, ensure a barrier separates the sample from the pack; crushed ice transportation can directly contact the sample. Secure the sample tube to prevent transport-related agitation, and deliver it to the laboratory within 24 h.

• Other Liquids

For other liquids, directly extract and place them in a 50 ml centrifuge tube, stored at 4°C. It is advisable to microscopically examine a small liquid sample to observe the cell state and estimate the total cell count, ensuring an adequate quantity for subsequent formal experiments. Follow the same transportation guidelines as for blood samples. Given the diversity of sample types, it is recommended to consult our technical team before proceeding.

Cell Samples

• Cultured Cells

Sample Preparation: Cultured cells must undergo microscopic examination to ensure their optimal state, free from contamination. The total cell count should exceed 10⁶. Culture flasks should be filled with the appropriate medium, and their openings sealed with film.

Shipping: Room temperature transport is recommended. During transit, carefully wrap the sample in ample bubble wrap to prevent damage.

• Freezing Cells

Sample Preparation: Prepare the freezing medium (e.g., 90% FBS + 10% DMSO). Equilibrate the cell freezing box at 4°C. Collect, count, and centrifuge cells, discarding the supernatant. Resuspend cells in the freezing medium (1 mL per 2×106 cells), ensuring thorough mixing. Distribute the cell suspension into -20°C pre-equilibrated tubes, then transfer to a -80°C freezer. If a gradient freezing box is unavailable, place cells at 4°C for 30 min, -20°C for 30 min, and finally to -80°C. Alternatively, commercial cell freezing solutions may be employed following their specific guidelines.

Shipping: Utilize dry ice for delivery. Ensure an adequate supply of dry ice, considering regional and weather variations, to prevent insufficient delivery that could compromise subsequent experiments.

• Plant Cells

For single-cell sequencing projects involving plants, our preference is to receive live plant samples directly. This is because the successful dissociation of protoplasts necessitates an optimal level of plant growth status. For tailored projects and specific requirements, we encourage you to consult with our technical team for expert guidance.

Sample for Spatial Transcriptomics 

For spatial transcriptome projects, it is recommended to directly perform OCT embedding on fresh or plant tissues, followed by freezing and storage at -80°C. Subsequently, send the samples to our laboratory on dry ice.

• Sample Preparation
1. Prepare crushed ice and dry ice.
2. Pre-cool OCT on crushed ice for 30 minutes.
3. Select an appropriate-sized embedding mold for tissue samples.
4. Use laboratory paper towels to absorb excess blood or liquid on the tissue surface to prevent ice crystal formation.
5. Take photos to confirm tissue orientation.
6. Wrap tissues directly with pre-cooled OCT, avoiding the formation of air bubbles.
7. Transfer wrapped tissues to pre-cooled molds on crushed ice, continuously adding OCT to ensure submersion.
8. Mark the orientation on the mold.
9. Place the tissue in dry ice until OCT freezes completely white.
10. Remove the frozen tissue block from dry ice, seal it, and keep it in -80°C for storage.
• Shipping

Utilize dry ice for delivery. Ensure an adequate supply of dry ice considering regional or weather variations to prevent insufficient delivery, which might impact subsequent experiments.

FFPE Samples

• FFPE Spatial Transcriptomics

We currently accept paraffin-embedded tissue samples of human and mouse. Various sample forms can be delivered, including paraffin-embedded blocks, labeled white slices, and H&E-stained slices (10-15 slices of 4-5 μm thickness). Before formal experiments, quality check the samples (H&E staining, DV200 should be more than 30%). Subsequent experiments can proceed once the samples meet the requirements.

• FFPE Single Cell Samples

For single-cell transcriptome sequencing of FFPE samples, utilize the 10X Fixed RNA Kit. We accept paraffin-embedded blocks or paraffin rolls of human and mouse FFPE samples for this purpose.