Single-Cell 3′ RNA Sequencing

Single-Cell 3′ RNA Sequencing

Service Description

Single cell RNA-Seq enables transcriptome heterogeneity study at the resolution of a single cell. It also unlocks the spatial-temporal gene expression profile with cell-by-cell resolution, which is unarchivable by standard, bulk RNA sequencing. At BGI we provide high-throughput single cell gene expression profiling service using 10x Genomics Chromium Single Cell Platform to unveil 3’ end counting of mRNA transcripts. Our turn-key solution (cell/tissue-to-report) and premade library service (library-to-data) for Single cell RNA-Seq provide flexible tool set for you depending on your preference, equipment accessibility and schedule.

BGI’s Single Cell Sequencing Solution utilizes the 10x Genomics® Chromium™ system for sample prep and library construction. Sequencing of the single cell libraries can be conducted on our proprietary DNBSEQ™ platform to ensure the downstream service quality, or the Illumina® platform (HiSeq and NovaSeq) by customer’s preference. BGI also provides sequencing services for customer self-prepared libraries using 10x Genomics® Chromium™ system Single Cell 3′ Library Construction Kit v3 and Next GEM Single Cell 3’ Library Construction Kit v3.1.

Applications

Sequencing Technology

Sequencing using DNBSEQ™ (DNA nanoball technology) is accomplished through RCR (rolling circle replication), a linear amplification that each copy is amplified independently using the same single strand circle (DNA nanoball, DNB) as template. As a result, error reads will not accumulate, and most of the signal originate from correct indices [1]. This enables elimination of index mis-assignment which is the major cause for the index hopping problem on Illumina® sequencing platform [5].

BGISEQ human whole genome sequencing

With our extensive experience in single cell sequencing we offer the highest quality services in sample prep, library construction, DNB conversion, sequencing, and bioinformatics analysis to our customers. The high-quality results comparing with the competitors have been published in leading scientific journals [2] [3] [4].

Project Workflow

Highly experienced laboratory professionals follow strict quality procedures to ensure the integrity of your results.

We employ rigorous QC steps following each phase of the service workflow as described by the flow chart below. The qualified sequencing data will be offered for download and bioinformatics service is available upon request.

How to order

We care for your samples from the start through to the result reporting. Highly experienced laboratory professionals follow strict quality procedures to ensure the integrity of your results.

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Send us a no obligation request for quote and our dedicated sales team will reply to you within 24 hours.

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References

1. Reliable Multiplex Sequencing with Rare Index Mis-Assignment on DNB-Based NGS Platform
https://bmcgenomics.biomedcentral.com/articles/10.1186/s12864-019-5569-5
2. Single-cell Transcriptomic Landscape of Nucleated Cells in Umbilical Cord Blood. bioRxiv 346106 (2018). doi:10.1101/346106 https://doi.org/10.1101/346106
3. Comparative analysis of sequencing technologies for single-cell transcriptomics. Genome Biology, volume 20, Article number: 70 (2019) https://genomebiology.biomedcentral.com/articles/10.1186/s13059-019-1676-5
4. Comparative performance of the BGI and Illumina sequencing technology for single-cell RNA sequencing. bioRxiv 552588 (2019). doi:10.1101/552588 https://www.biorxiv.org/content/10.1101/552588v2
5.Effects of Index Misassignment on Multiplexing and Downstream Analysis (Illumina white paper, 2017).

 

Sequencing Service Specification

BGI Single Cell sequencing services are executed with the DNBseq sequencing technology, featuring cPAS and DNA Nanoballs (DNB™) technology for superior data quality.

  • Sample preparation and services

    Sample preparation and services

    • 1X10^6 fresh frozen cells/100 mg fresh frozen tissue per sample submission
    • PE 100 sequencing is offered
    • 20K read pairs/cell recommended
    • Raw data and bioinformatics analysis are available in standard file formats
    • Advanced and custom bioinformatics data analysis
    • Cloud-based data storage and delivery system
    • Standard single cell 3' RNA seq data analysis is available
    • Customized data analysis is available
  • Sequencing Quality Standard

    Sequencing Quality Standard

    • Guaranteed ≥80% of bases with quality score of ≥Q30
  • Turn Around Time

    Turn Around Time

    • Single Cell 3’ RNA Sequencing Solution (tissue/cell-to-report) -- Guaranteed 6 weeks from tissue/cell QC acceptance to filtered RAW data availability.
    • Single Cell 3’ RNA self-prepared libraries sequencing (library-to-data) -- Guaranteed 4 weeks from library QC acceptance to filtered RAW data availability
    • Rapid turnaround time of 2 weeks is available with qualified sample volume. Contact your local BGI specialist for details

Sample Requirements

Cell line submission

BGI recommends fresh-frozen cells to be submitted for this procedure, however we do provide service for cryopreserved cells. Please follow the below guidelines for cell submission.

Place 5×105 to 1×106 cells in 1 ml of freezing media per cryotube and place the tube in gradient freezing container with isopropyl alcohol at room temperature, move the container to -80°C and keep freezing overnight.

The standard freezing media component is cell growth media + 20% FBS + 10% DMSO; or 90% FBS + 10% DMSO.

For detailed cell preparation guideline download document: Cell Line Preparation Guideline

 

PBMC submission

BGI recommends fresh-frozen PBMC to be submitted for this procedure, however we do provide service for cryopreserved cells. Please follow the below guidelines for cell submission.

For detailed PBMC sample preparation guideline download document: PBMC Preparation Guideline

 

Tissue submission

BGI provides the service for the tissue which has been frozen as whole prior to the dissociation step. Due to this more challenging, non-standard service workflow, extra steps such as the control tissue dissociation and dead cell removal may be required. This also leads to extra time and cost for this service.

The requirements for the fresh-frozen tissue submission:

Minimum 100 mg gradient cryopreserved fresh tissue/sample

For detailed tissue sample preparation guideline download document: Tissue Sample Preparation Guideline

 

Premade library submission

BGI also provides sequencing services for customer self-prepared libraries using 10x Genomics® Chromium™ system Single Cell 3′ Library Construction Kit v3 and Next GEM Single Cell 3’ Library Construction Kit v3.1, that meet the following requirements:

Library parameter Requirement Preferred QC method
Fragment size 400bp±100bp Agilent 2100
Concentration ≥2ng/ul Qubit
Total amount ≥30 ng Qubit

Once the sample is ready, please contact your local BGI specialist for dry ice shipping arrangement.

Data Analysis

Standard Anlysis for single cell RNA sequencing

  • Sequencing data QC

  • Data mapping

  • Quantitative analysis

  • Cell cluster analysis

  • Differentially expressed genes identification

  • Marker genetic identification

  • Ontology analysis of Differentially Expressed Genes (DEGs)

  • Pathway analysis of DEGs

  • Transcription factor (TF) coding ability prediction of DEGs

  • Protein interaction analysis of DEGs

  • Genetic correlation network analysis

Customized analysis

In addition to raw data output, BGI offers a range of standard and customized bioinformatics pipelines for your single cell sequencing project.

Single Cell DNA/RNA Sequencing Resources

We care for your samples from the start through to the result reporting. Highly experienced laboratory professionals follow strict quality procedures to ensure the integrity of your results.

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