Library Preparation with cellenONE

 

cellenONE provides high accuracy single cell isolation but it also present a number of additional features to miniaturise library preparation

  • Nanoliter Dispensing

    cellenONE allows high precision dispensing of any volume of reagents from 100pL to several µL
     

  • High Precision Positioning

    cellenONE uses high resolution robotics allowing dispensing of reagents in any substrates 96, 384, 1536, 3456 microwell plates as well as nanowell substrates (e.g. cellenCHIP™)

  • Environmental Control

    cellenONE offers advanced environmental control with temperature, humidity and automated dew point controls which are essential features when working with sub microliter volumes
     

  • Multiple Interventions

    Unlike microdroplet-based methods where multiple reagents additions are impossible, cellenONE allows multiple intervention which permits implementation of most single cell omics methods
     

Reagent addition in low volumes: (Lysis buffer + RT mix) addition (40nL/well).
Credits: Derek Bogdanoff. UCSF. 2017

BENEFITS

50x to 100x Reagent Volume Reduction 

(vs. 96/384wp)

Drastic Costs Reduction

Implementation of Single Cell Omics Methods 

Lower Background and Better Sensitivities

 

 

  • scYour-Seq

    By combining high accuracy single cell isolation with nanoliter dispensing capabilities, a myriad of molecular biology and proteomic protocols can now be miniaturized using cellenONE.

    If you have established or novel protocols you’d like to miniaturized, and would like to discover how to implement those using cellenONE, please don’t hesitate contacting us at contact@cellenion.com
     

  • Barcoding Services

    Cellenion offers a range of barcoding services to facilitate implementation of your protocols into cellenCHIP or other nanowell substrates.

    Both solution-based oligo barcodes (e.g. oligodT barcodes) or barcoded beads can be preloaded in the substrate of your choice.

    Contact us at contact@cellenion.com to discover more.
     


A success story: DLP+

 

By combining cellenONE’s single cell isolation and nanoliter dispensing features, the groups of Sam Aparicio (UBC, Vancouver) and Sohrab Shah (MSKCC, New York) have developed a scalable single cell whole genome sequencing method called Direct Library Preparation Plus (DLP+).

Details of this method was recently published as an open-access article in the journal Cell.

Results generated have been compiled into a web-based interactive visualisation platform (www.cellmine.org).
 


This affordable, low bias method allows in depth analyses of a variety of samples.

It allows determination of copy number variation (CNV), single nucleotide polymorphysim (SNP), cluster analyses and phylogenic reconstructions.

This method is now implemented as a routine at the British Columbia Cancer Research center and being adopted by leading research centers worldwide.
 

 

Method developed by

BENEFITS

High quality single cell whole genome libraries

CNV, SNP, Cluster analyses and Lineage reconstruction

Any cells and nuclei from 5 to 80μm

Low reagent costs (0,27 US$/cell) (ref: BioRxiv)

This method is now implemented at these world leading research centres


Low Cost scRNA-seq Libraries with cellenCHIP™

 

To successfully miniaturize library preparation, it is essential to use substrates that have been designed for sub-microliter volume work.  

Cellenion has developed cellenCHIP™, an innovative nanowell chip containing 384 wells on a microscope slide footprint.

In total, up to 8 cellenCHIPs (3072 single cells) can be placed on the cellenONE deck.  

 

If you’d like miniaturize your library preparation method onto cellenCHIP, please contact us and enrol in our early technology adopter program (ETAP)


Commercial Kit Miniaturization in cellenCHIP

 

cellenONE and cellenCHIP were used to miniaturize a commercial 96wp-based transcriptomic kit (QIAseq UPX 3', Qiagen).

All concentrations used were kept identical to manufacturer’s recommendations. 

By miniaturizing reaction volumes to 100nL per well, it was possible to prepare libraries for 1536 single cells (4x cellenCHIPs) from only one 96wp kit.

Results showed equivalent performances (UMIs and number of detected genes /single cell) in cellenCHIP and 96wp!
 

QIAseq UPX 3' cellenCHIP
96 unique oligo dT 96 unique oligo dT
5 μl/well 100 nL/well
96 single cells 1536 single cells
€27/cell €2.5/cell

The following steps describe the workflow using QIAGEN kit, cellenCHIP™ and cellenONE®

 

 

 

 

BENEFITS

Data quantitatively aligns with other scRNA-seq technologies

Seamless implementation of a commercial 96wp kit into cellenCHIP.

Miniaturization of lysis and reverse transcription in barcoded cellenCHIP

Equivalent to higher performances compared to 96wp format.

Drastic reduction of costs from 27 € to less than 2.5 € per single cell
 


Homebrew 3’ scRNA-Seq in cellenCHIP

 

A 3’ scRNA-Seq protocol was developed in cellenCHIP using a range of commercially available reagents.

The following steps describe the workflow using cellenCHIP and cellenONE®

 

3 different reverse transcriptase were compared (RTase_1 to _3).

To validate these protocols, two cell types (HEK and NIH_3T3) were sequentially isolated as a checkerboard pattern together with several controls.

OligodT barcoded wells were filled with 100nL of a buffer mix containing (lysis and RT reagents).
 
After single cells isolation, cellenCHIP were sealed and placed into a thermocycler for reverse transcription and template switching reactions.

cellenFUNNELS were used for pooling reaction mixture into microcentrifuge tubes and remaining steps (cDNA amplification, tagmentation and library amplification) were carried out as described in the literature.

After sequencing, and bioinformatic analyses all 3 enzymes showed reproducible results.

RTase_1 performed best with up to 7000 detected genes per single cell with a median of 5000 detected genes per single cell.
 

All libraries were analysed (Bioanalyzer) and consistently displayed fragments of about 500bp.
 

BENEFITS

Compatible with a range of commercially available enzymes and reagents. 

Method suited for various samples size from few (<100) to 1000’s cells.

Simplified protocol with combined lysis and RT in 100nL in cellenCHIP.

Low number of PCR cycles (<18) to minimize data bias, and limit UMI replicates.

High reproducibility observed within biological replicates.

High quality with minimal to null background.

High performance with up to 7000 genes detected per single cell (median 5000 genes detected) with a low quantity of reads needed (median 100,000 mapped reads per single cell).