Platform foR SinglE Cell GenomIcS and Epigenomics (PRECISE)

Single cell genomics is entirely changing the way how we are characterizing cellular responses in homeostasis and many common diseases. In fact, the cellular landscapes will be re-written for many complex organs and diseases.

Platform for Single Cell Genomics and Epigenomics

The "Platform for Single Cell Genomics and Epigenomics" is a joint Venture between the University of Bonn and the German Center for Neurodegenerative Diseases (DZNE). Both institutions seek to conjointly increase their expert knowledge in the areas of genomics and bioinformatics. Particular focus lies on the development and application of tools and technologies for single-cell genomics and on providing these technologies for internal and external collaborators.

The laboratory space of the platform is situated in the new DZNE building B, the bioinformatics unit is located at the LIMES Institute. Both units are closely collaborating.

Some of the technologies offered at PRECISE are listed below:

SMART-seq2 (Picelli et al., 2013, Nat Methods) is the single cell RNA-seq (scRNA-seq) method with the highest sensitivity. It allows sequencing of the transcriptomes of hundreds of cells in parallel, over the entire transcript length  and entirely relies on off-the-shelf reagents. SMART-seq2 is the best choice when it comes to the study of splice variants, SNPs or monoallelic gene expression. Library preparation costs are significantly reduced by using an in-house version of Tn5 transposase (Picelli et al., 2014, Genome Research).

FLASH-seq is an innovative full-length single-cell RNA sequencing method with high sensitivity that necessitates minimal manual intervention, as the protocol can be performed automation-friendly from lysed cells to pooled cDNA libraries in less than five hours (Hahaut et al., 2021, Nat Biotech). 

Simultaneous electrophysiological, transcriptomic and morphologic profiling of single cells (Cadwell et al., 2016, Nat Biotech) based on the patch clamp technique and SMART-seq2 method.

PRECISE was the only alpha-tester in Europe for this technology, recently introduced on the market by BD Genomics. Rhapsody is based on the CytoSeq method (Fan et al., 2015, Science) and allows the 3´-end sequencing of up to 50K cells/experiment with targeted (partly) customizable gene panels (500 or 1000 genes) or of the whole transcriptome. Rhapsody uses microwell arrays for single-cell capture and is equipped with an imaging system for cell visualization and counting. The price for sequencing of a targeted panel is considerably lower since it doesn´t require the analysis of the entire transcriptome. Rhapsody samples can be multiplexed and/or surface proteins can be simultaneously quantified during RNA-Seq based on the CITE-Seq method (Stoeckius et al., 2017, Nat Methods).

The commercial emulsion droplet method of 10x Genomics allows to analyze tens of thousands of cells in parallel in a robust and reproducible way (Zilionis et al., 2017, Nat Protoc).

Single-cell transcriptomics is a rapid moving field, where new library preparation and computational methods are introduced every few weeks. It is therefore vital to keep up with the pace of technological development. We are investing important amounts of time, human and technical resources in testing and implementing recently released new single-cell technologies as well as developing better, faster and inexpensive methods to be able to offer the most cutting-edge technologies available and tackle ever more complex biological questions.

Some of the new technologies in the pipeline are listed below:

 To characterize open chromatin genomic regions, a proxy for finding transcriptionally active gene regions.

A “split-and-pool”  3´-sequencing method based on an approach that makes use of combinatorial indexing and Tn5 transposase (Cao et al., 2017, Science). SciRNA-seq works on fixed cells, does not require single cell sorting and is easily scalable to thousands of cells, thus reducing the per cell library preparation cost.

A method capable of isolating RNA from fixed, permeabilized, stained, and sorted cells which is later converted to sequencing library with the Smart-seq2 method. Currently the method works with >400 cells (Thomsen et al., 2016, Nat Methods).

High-precision long-read sequencing (also known as HiFi sequencing) enables full-length RNA sequencing and helps to identify RNA isoforms that may be important in the development of neurodegenerative diseases or to elucidate biological functions. HiFi sequencing leads to more information and new applications such as full-length mRNA sequencing, discovery of new isoforms, comprehensive variant detection or targeted sequencing at the single-cell level (Healey et al., 2022, Genetics). 


Equipment of the platform

Liquid handling and QC systems

  • BioNex Nanodrop II
  • Dispendix I.Dot
  • SPT Labtech Mosquito x1
  • Tecan Freedom EVO
  • Roche Lightcycler 480
  • Agilent Tapestation 4200
  • Agilent Fragment Analyzer
  • Thermo Fisher Qubit 3, Flex

NGS library generation

  • BD Rhapsody HT Xpress, Scanner and Station
  • 10X Genomics Chromium and Chromium iX
  • Cytena C.Wash
  • Covaris LE220
  • Diagenode IP-Star
  • Pippin Prep Instrument

Sequencing instruments

  • Illumina MiSeq
  • Illumina NextSeq 2000
  • Illumina NovaSeq 6000
  • Illumina NovaSeq X Plus
  • PacBio Sequel II

Spatial transcriptomics

  • Nanostring GeoMx Digital Spatial Profiler
  • 10X Xenium Analyzer

 

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