Available Services

The NF for Genomics will provide sequencing of pools of libraries prepared by the users with the MiSeq sequencing platform (Illumina).

The MiSeq, an Illumina platform, is a compact benchtop sequencer ideal for smaller-scale sequencing projects. It employs sequencing-by-synthesis technology, enabling the determination of DNA sequences via fluorescently labeled nucleotides. With compatibility with various reagent kits and chemistries, users can tailor sequencing protocols to their experiment needs. Commonly used for targeted sequencing (amplicon sequencing, target capture, and custom panels), small genome sequencing (bacterial or viral), metagenomics, 16S rRNA sequencing, small RNA-Seq for gene expression profiling, and viral genome sequencing, the MiSeq facilitates diverse applications in genomics research, particularly in microbial diversity studies and viral evolution analysis.

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iPSCs are generated from Peripheral Blood Mononuclear Cells (PBMCs) using footprint-free reprogramming Sendai virus technology.

Every iPSC line undergoes thorough testing to ensure quality and consistency:

• Cell identity confirmation using STR analysis
• Sendai virus clearance
• Expression of undifferentiated stem cell markers
• Assessment of pluripotency markers through 3-germ layer differentiation assay
• Karyotyping (Q-banding) and identification of Copy Number Variations (CNVs) at high resolution
• Master bank post-thaw viability and mycoplasma testing.

Both live and frozen purified PBMCs qualify for the service.

The service typically requires 3 to 4 months for completion and includes the delivery of 1-3 clones, each provided in 10 cryopreserved vials.

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Fibroblasts are reprogrammed using footprint-free, non-modified RNAs (nm-RNA).

Every iPSC line undergoes thorough testing to ensure quality and consistency:

• Cell identity confirmation using STR analysis
• Sendai virus clearance
• Expression of undifferentiated stem cell markers
• Assessment of pluripotency markers upon 3-germ layer differentiation assay
• Karyotyping (Q-banding) and identification of Copy Number Variations (CNVs) at high resolution
• Master bank post-thaw viability and mycoplasma testing.

Both live and frozen fibroblasts at a low passage number (maximum passages 5) qualify for the service.

The service typically requires 3 to 4 months for completion and includes the delivery of 1-3 clones, each provided in 10 cryopreserved vials.

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The service offers dedicated pipelines for small- and large-scale cell cultures and expressions:

• Small scale: 15 litres fermenter useful for initial trial test before moving to larger scale fermenter or small expression using both bacteria and yeast cells. Typical culturing strategies are available such as batch and fed batch (up to 2 feeds). Max working volume of 10 litres.
• Large scale: 150 litres fermenter dedicated to large cultivation and protein production. Typical cultivation strategies are available, including batch and fed batch (up to 2 feeds). Max working volume of 100 litres.

In both cases, cells will be collected and separated from the supernatant using dedicated superspeed centrifuges or a High-Speed Tubular Centrifuge. Upon request, the cells can be lysed using a continuous flow cell disruptor or by a high-capacity cryogenic grinder.

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The service offers dedicated pipelines for small- and large-scale cell cultivation and protein expressions:

• Small scale: utilizing commercial Expi293F cells, transient transfection is conducted with a plasmid containing the gene\s of interest (potentially with a fluorescent marker), provided by the applicant. Cell cultivation and transfection take place in Erlenmeyer flasks within dedicated shaker incubators; typically, 500 ml of cell culture per 2-liter flask is utilized up to a total cultivation volume of approximatively 10 litres. Transfection of an Expi293F cell culture is performed at approximately 2 to 3 x10^6 cells\ml, following a modified protocol with polyethylenimine (PEI). While no expression test will be performed, the efficiency of transfection can be readily assessed if the plasmid includes a fluorescent marker. Transfected cells are subsequently harvested by centrifugation, and either the frozen pellet or the refrigerated supernatant will be provided.
• Large scale: bioreactor cultivation and protein expression are performed either with commercial cell lines (e.g., Expi293F cells) transiently transfected with applicant-provided plasmids, or with any mammalian cell line adapted to suspension cultivation supplied by the applicant. Preliminary tests are normally performed using a 0.5 litres scale bioreactor to define and optimize bioreactor cultivation conditions. Subsequently, a 10 litres scale bioreactor enables large-scale cell cultivation and protein expression, employing both transient transfection protocols and stable cell line expressions. Moreover, the implementation of perfusion, utilizing an acoustic retention device, may be explored to increase the biomass production and, consequently, protein expression. Similar to small-scale operations, no expression test will be performed. Instead, monitoring of transfection efficiency for transient expression can be undertaken if the plasmid carries a fluorescent marker or fluorescently tagged protein of interest.

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The service aims to cover the entire process of Baculovirus protein expression, starting from a Bacmid. Initially, commercial Sf9 cells are transfected with the Bacmid, supplied by the applicant, which contains the gene of interest and a fluorescence marker. The initial stage results in a low-titre Baculovirus suspension (P1 generation), followed by a second amplification (P2 generation) to produce a high-titre suspension. The P2 Baculovirus suspension could be directly used for protein expression or another round of Baculovirus amplification is performed before infecting commercial High Five insect cells (at 0.5×10^6 cells\ml). The virus amount used depends on the construct and might need optimization in initial small-scale infections. Protein expression is monitored indirectly by checking for the fluorescence marker (carried by the Bacmid) and cell viability. Finally, cells are harvested via centrifugation, and the pellet or supernatant is made available.

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Spinning disk confocal microscope with 7 excitation laser lines (405 nm, 446 nm, 477 nm, 520 nm, 547 nm, 638 nm, and 749 nm), 4 high-power laser lines for FRAP and TIRF (405 nm, 488 nm, 520 nm, and 640 nm), 4 high-speed, large FOV back-illuminated sCMOS cameras.

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We offer sample preparation by negative stain EM followed by TEM imaging at 120 kV. A maximum of 1 specimen and 8 grids can be prepared and processed per unit of service. Imaging will be provided for a maximum of 8 continuous hours per unit of service. An optional “polishing” size-exclusion chromatography (SEC) step can be performed on thawed material by the Biophysics Unit, as and if specified in the request for this service. 400 mesh copper grids with amorphous carbon film layer will be used as support. Glow discharging will be performed by a Pelco EasyGlow device. Staining will be performed with a 2% (w\v) uranyl acetate aqueous solution. Imaging in TEM mode at 120 kV will be performed on a Thermo Scientific Talos L120C equipped with CETA 16M camera. Imaging conditions requested by the User, if provided, must be compatible with Facility practices. This service will only be performed by Facility Staff.

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The native barcoding kit enhances nanopore sequencing by enabling simultaneous sequencing of multiple samples with unique barcodes. After gDNA extraction, each sample is tagged with a barcode during library preparation. Sequencing generates long reads, allowing real-time base calling. Bioinformatics tools can be used to analyze data, including aligning reads and variant calling. This approach is particularly useful for studying small bacteria genomes because it is cost-effective and efficient, beneficial for microbiome studies and environmental monitoring and other applications in microbial genomics.

Libraries are prepared with the Native Barcoding Kit 96 V14 that enables PCR-free multiplexing of small gDNA samples using 96 unique barcodes. It involves repairing and dA-tailing gDNA, then ligating a unique dT-tailed barcode adapter. Barcoded samples are pooled, and each barcode adapter ligates to a sequencing adapter. Optimized for high sequencing accuracies (>99% Q20+) on nanopore Flowcells R10.4.1.

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Nanopore sequencing directly sequences DNA using nanopores, known for producing long or ultra-long reads. Protocol includes DNA extraction, minimal fragmentation for long reads or no fragmentation for ultra-long reads generation, DNA end repair, adapter ligation, device setup, sequencing, and data collection through electrical signals. Base-calling converts signals into DNA sequence. Data analysis involves error correction, read filtering, and read assembly.

The library prep method for long-read sequencing involves repairing DNA ends, dA-tailing, and ligating sequencing adapters. It achieves >99% sequencing accuracy (Q20+) on R10.4.1 flow cells. It is compatible with target enrichment, whole genome amplification, and size selection.

The Ultra-Long DNA Sequencing library prep method facilitates preparation of ultra-high molecular weight DNA, yielding N50s >50 kb and reads up to 4+ Mb. Utilizing transposase chemistry, it cleaves template molecules and attaches tags, followed by rapid adapter addition.

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