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TEST PAGE 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 determine and thoroughly evaluate a cell or nucleus isolation strategy suitable for their target tissue and research question. Users are furthermore strongly encouraged to evaluate their sample quality (e.g. RNA integrity) preceding 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.

Available services

 

  scGenome scTransciptome scEpigenome
Takara iCell8 cx d ✓ d
10X Genomics ✓ d ✓
cells d ✓ d
nuclei d ✓ ✓
cell surface protein   ✓ d
multiplexing   ✓ d
CRISPR guides   ✓  
targeted TCR/BCR   ✓  

✓ available protocol
d in development

10x Genomics Chromium system
 

The Chromium system enables the user to analyze up to several thousand 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 of up to and over 2000 genes per cell. 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 partially as well as to combine several low-input samples in one reaction. Therefore, the 10x protocol for 3´mRNA can be combined with:

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

CITE-seq and Feature Barcode provide access to similar information on cell surface protein and enable hashing. Feature Barcode tags are captured via the capture sequence 1 on the 10X 3'mRNA beads and cell surface protein and hashing tags will be combined in one library. In TotalSeqA-based CITE-seq, antibody tags are captured identically to mRNA via their polyA-tail. 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 TotalSeqB and Feature Barcode Technology.

 
Possible combinations CiteSeq/TotalSeq A Feature Barcode/TotalSeq B Cell Plex CRISPR
CiteSeq/TotalSeq A ✓   p d
Feature Barcode/TotalSeq B   ✓ p d
Cell Plex p ✓ ✓ ✓
CRISPR d p ✓ ✓

✓ available protocol 
d  in development
p  possible

Single-cell Immune Profiling library preparation (v2, sc Immune profiling)

High throughput single cell analysis for B- or T-cells from human or mouse (several 1000s of cells per library). 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, 10X Genomics - scATAC)

Suitable for high throughput epigenomic analysis (several 1000s of nuclei per library) at single cell resolution to infer cellular heterogeneity based on accessible chromatin structures.

Single-cell Multiome ATAC + Cell Gene Expression (10X Genomics - scMultiome)

Combines the ATAC and 3’mRNA workflow from 10X Genomics. With the Multiome kit it is possible to simunanously profile open chromatin and 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.  

TakaraBio iCell8cx
 

Smart-Seq for full-length mRNA-seq library preparation using the iCell8cx (Takarabio - SMARTer)

Medium throughput (up to 1500) single cell transcriptome analysis providing high gene detection (up to 10 000 genes per cell) as well as accessing the entire length of captured transcripts. Suitable to study for example splice variants or gene fusions. Imaging combined with live-dead staining provides stringent quality control to strictly exclude dead and/ or multiple cells from downstream processing and analysis.

Next-Generation Sequencing

Libraries are sequenced using a NovaSeq 6000 or a NextSeq 500 (Illumina).

Computational support
 

All 10x Chromium-based services include a final report containing a Cellranger 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 as well as aggregated data.  

For full-length mRNA sequencing on 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 deposited to be included during sequencing read alignment.

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

24 Jun 2021