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Single-Cell Genomics Platform

Since 2019 CTG offers a dynamic, state-of-the-art platform for single-cell sequencing to users at Lund University with the aim to provide expertise, accessibility, proximity and service to researchers in need for single-cell genomics.

General guidelines

Gentle sample isolation is essential not to bias against vulnerable cell types while fast isolation is desirable to reduce degradation of nucleic acids.
Thus, prior to initiating a project at the Single-Cell Genomics Platform, users are asked to thoroughly evaluate a cell or nucleus isolation strategy suitable for their target tissue and research question. Users are also strongly encouraged to evaluate their sample quality (e.g. RNA integrity) prior to submitting single-cell genomics work at the facility.
Once a project is accepted by the Single-Cell Genomics Platform, users must follow sample preparation and deposition instructions provided by the platform.

Requirements to start a single-cell project

In order to start a single-cell project with us, we want to confirm that your isolation protocol is successful. Therefore, you must submit the following information prior to project start:

For single-cell suspensions:

  • image of cell suspension (absence of debris and clumps)
  • viability staining (optimal >90%, acceptable >70%)
  • cell count (manual or automatic, no FACS count)

You are furthermore strongly encouraged to evaluate you sample quality (e.g. RIN value).

For single-nucleus suspensions:

  • image of nuclei suspension (absence of debris, no blebbing, well defined membrane)
  • nuclei count (manual or automatic, no FACS count)

Available services

Tabell. Single Cell

10x Genomics Chromium system

The Chromium system enables the analysis of several thousands of cells or nuclei per library. We offer library preparations for various single-cell applications:

  • Single-cell Gene Expression (3´GEX) library preparation (v3.1, sc mRNAseq). Suitable for high throughput single-cell transcriptome analysis (several 1000s of cells or nuclei per library) with detection in the range of 1500-6000 genes per cell, depending on cell type. Full-length mRNA information is not preserved.

For bimodal single-cell analysis, it is possible to combine transcriptome analysis with the expression analysis of cell surface markers (pre-defined by user) or the detection of CRISPR guide RNAs. Cell hashing may be useful to compensate for technical variability between simultaneously processed samples. It  can also be used to combine several low-input samples in one reaction. Therefore, the 10x protocol for 3´mRNA can be combined with:

  • CITE-seq (TotalSeq A antibodies)
  • Cell Hashing (TotalSeq A antibodies)
  • Feature Barcode Technology (10x Genomics):
              Cell Surface Protein (TotalSeq B antibodies)
              Multiplexing (Cell Plex or TotalSeq B antibodies)
              CRISPR Screen

Both CITE-seq and Feature Barcode Technology provide access to similar information on cell surface proteins and enable hashing. Feature Barcode tags (TotalSeq B) are captured via the capture sequence 1 on the 10x 3'mRNA beads, therefore cell surface protein and hashing tags are combined in one library. In TotalSeq A-based CITE-seq, antibody tags are captured identically to mRNA via their polyA-tails. Cell surface protein barcodes (ADT) and hashing tags (HTO) are subsequently generated as separate sequencing libraries. This in turn enables sequencing of ADT and HTO at different read depths, otherwise not possible when using TotalSeq B and Feature Barcode Technology. Only Feature Barcode is supported by 10X Genomics.

Tabell 2. Single cell
  • Single-cell Immune Profiling library preparation (v2, sc Immune profiling). High throughput single-cell analysis of B- or T-cells from human or mouse (several 1000s of cells per library) samples. Combines 5’mRNA transcriptome analysis (5´GEX) with full-length sequences for immune receptors (BCR/TCR) and expression analysis of cell surface proteins through Feature Barcoding. Full-length mRNA information is not preserved. Can be combined with Feature Barcode Technology to capture Cell Surface Protein and for Cell Hashing (TotalSeq C).
  • Single-cell ATAC-seq library preparation (v1.1,  scATAC). Suitable for high throughput epigenomic analysis (several 1000s of nuclei per library) at single-cell resolution to infer cellular heterogeneity based on transposase-accessible chromatin structures.
  • Single-cell Multiome ATAC + Cell Gene Expression (scMultiome) Integrates the ATAC and 3’mRNA workflow from 10x Genomics. With the Multiome kit it is possible to simunanously profile open chromatin and the transcriptome of a single cell, while processing up to several 1000s of nuclei in parallel. This kit cannot be used in combination with CITESeq, Cell Hashing or the Feature Barcode Technology at present.  

iCell8cx system

  • Smart-Seq for full-length mRNA-seq library preparation using the iCell8cx in combination with the Takara Smart-seq Pro chemistry. Medium throughput (up to 1500) single-cell or -nucleus transcriptome analysis. Provides high gene detection (up to 12 000 genes per cell) as well as sequence information for the entire length of captured transcripts. Suitable to study splice variants or gene fusions. Imaging of cell-per-indexed-well combined with live-dead staining provides stringent quality control to strictly exclude dead cells and/or multiplets  from downstream processing and analysis.
  • Whole-genome sequencing based on direct library preparation plus (DLP+) introduced by Laks et al. 2019 and adapted to the iCell8cx. This approach enables the analysis of copy number variation at single-cell resolution in up to 1500 individual cells per experiment. On-chip imaging analysis enables stringent quality control and to discriminate between singlets and multiplets.  

Next-Generation Sequencing

Libraries are sequenced using a NovaSeq6000 or a NextSeq2000.

 
Computational support
All 10x Genomic services include a final report containing a Cell Ranger web summary file for quality control (QC) of the sequencing outcome, as well as FASTQ, BAM, gene count matrix containing all single cells or nuclei passing QC, basic visualization and clustering. The standard output also includes a .cloupe file for exploring your samples with the 10x Loupe browser for each sample individually as well as for multiple samples as aggregated data.  

For full-length mRNA sequencing on the iCell8cx, the final report will include a summary based on Takara’s Cogent NGS Discovery Software including information such as read and gene count per cell, ribosomal and mitochondrial reads per cell as well as initial cell clustering based on t-distributed stochastic neighbor embedding (t-SNE).

Usage of reporter genes need to be specified before project start and exact sequence information needs to be submitted to be included during sequencing read alignment.

For whole genome sequencing using DLP+ on the iCell8cx, the final report will contain a summary based on the DLP+ analysis pipeline providing information such as coverage depth, breadth and individual quality score per cell as well as chromosomal copy number information with single-cell resolution.

The standard computational service is free of charge and applies to studies carried out in mouse and human samples.

Applications under development

  • Single-cell mRNA-sequencing of fixed cells and nuclei using e.g. ParseBiosciences .
  • Combinatorial indexing on 10x Chromium for high throughput single-cell transcriptome analysis.
  • Single-cell Cut&Tag for iCell8cx. Profiling of epigenetic modifications at single cell resolution.
  • Combined Cell Surface Protein and CRISPR Screen detection for 3’ GEX v.3.1 (10x Genomics).


Once we have received the information stated above, we will get back to you within five workdays with a feedback on your request, including a feasibility assessment as well as information on pricing and sample deposition if applicable.

Contact

Development of protocols

If you are interested in collaborating with us for our protocols under development, or if you have a suggestion for the implementation or development of a novel method, please contact Ulrich Pfisterer (SingleCell [dot] CTGservice [at] med [dot] lu [dot] se).

Available services

To initiate a project using our available services, please start a project in iLab. ILab is a managing software for core facilities and increases the transparency of projects and quotes. It also includes a booking schedule for 10X Chromium Controller runs.

We have several user guides available. A quick guide for PIs to set up a group/lab, a starting guide for further lab members and a more detailed guide providing information on all functions in iLab. If you have any questions regarding iLab please write to CTGiLAB [at] med [dot] lu [dot] se.

Using iLab
You can login using your LucatID on the main Lund University iLab webpage.
You can also use this link, which brings you directly to our Service Request site after login.

Consultation and preliminary quotes
If you want to schedule a meeting to discuss your project or want to get a preliminary quote, choose “Consultation”, fill out the form, save it and click on “send request” at the bottom of the page. We will process your request as soon as possible.

Single-Cell Project
If you are ready to start a project click on “Single-Cell Project Request”, fill out the Contact & Invoicing Information and Single-Cell Project forms, save them both and click “send request” on the bottom of the page. We will check the forms and send you a quote.

 

 

Contact for service:

    SingleCell [dot] CTGservice [at] med [dot] lu [dot] se
    Phone: +46730968048

    Julia Bräunig
    Ulrich Pfisterer

    CTG Roll-up