Servizi disponibili

Point-scanning confocal microscope with multiphoton excitation and Airyscan2 detector, 5 laser lines (405 nm, 488 nm, 561 nm, 594 nm, and 639 nm), multiphoton excitation with a tunable Ti:Sa laser (700 nm – 1300 nm) and a fixed (1040 nm) laser line, 6 non-descanned detectors (4 PMT and 2 BiG), epifluorescence excitation and sCMOS camera.

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Point-scanning confocal microscope with Airyscan2 detector, 5 laser lines (405 nm, 488 nm, 561 nm, 594 nm, and 639 nm), sample finder, epifluorescence excitation and sCMOS camera.

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Lattice lightsheet with 3 lasers (488 nm, 561 nm, and 640 nm) and dual cameras.

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Lattice structured illumination and localization microscope with 4 laser lines (405 nm, 488 nm, 561 nm, and 638 nm), dual sCMOS cameras, TIRF, PALM, and STORM modules

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Binocular microscope with motorized stage and focus for large FOV acquisition, back-illuminated sCMOS camera and Apotome module.

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Manual, upright wide-field microscope with LED epifluorescence illuminator and monochrome sCMOS camera, condenser for phase and darkfield imaging.

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Automated slide scanner with color and monochrome cameras, LED epifluorescence illuminator with 7 LEDs (385 nm, 430 nm, 475 nm, 555 nm, 590 nm, 630 nm, and 735 nm), 6 objectives (2.5x/0.12, 5x/025, 10x/0.45, 20x/0.45, 20x/0.8, and 40x/0.95).

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Whole Genome Sequencing (WGS) is a robust methodology utilized in genomics research, personalized medicine, and clinical diagnostics. It entails determining the complete DNA sequence of an organism’s genome, providing understanding of genetic makeup and insights into genetic variations, evolutionary patterns, and disease mechanisms. WGS comprises: DNA extraction, library preparation involving fragmentation of DNA and addition of adapters, and sequencing using next-generation sequencing (NGS) platforms.

Bioinformatic analysis of WGS data involves assembling sequencing reads to reconstruct the genome sequence, annotating the genome, and calling variants to pinpoint differences between the sequenced genome and a reference. Illumina DNA PCR-Free library preparation protocol combines on-bead tagmentation and PCR-free chemistry. This approach ensures uniform coverage across the genome and is suitable for human WGS, de novo assembly of microbial genomes, and tumor–normal variant calling.

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Whole Exome Sequencing (WES) is a targeted approach focusing on the protein-coding regions of the genome (around 1-2%). The exome encompasses a significant portion of known disease-causing mutations, it is an affordable choice for various genetic studies where focusing on protein-coding regions suffices for research or clinical objectives.

WES begins with DNA extraction, followed by library preparation involving fragmenting DNA and adding adapters. Next is exome capture, where target-specific probes enrich DNA fragments corresponding to exonic regions, the prepared exome library undergoes high-throughput sequencing.

Bioinformatic analysis includes aligning reads to a reference genome and identifying genetic variants within exonic regions, such as single nucleotide variants (SNVs), insertions, deletions, and structural variations. In the NF Genomics protocol, TWIST Comprehensive Exome Panel is utilized, covering over 99% of protein-coding genes, with a design size of 41.2 Mb and targeting a total of 36.8 Mb, including an expanded content of RefSeq and GENCODE databases.

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This service is designed to cover imaging on large volumes and includes sample preparation by either high-pressure freezing or chemical fixation, freeze substitution, resin embedding, section preparation by ultramicrotomy and S\TEM imaging at 300kV. The User can provide specimen already fixed, stained, embedded as well as already sectioned and made ready for EM acquisition. A maximum of 1 specimen can be processed per unit of service, and 4 samples maximum (i.e., replicates) can be prepared per specimen. High-pressure freezing will be performed on a Leica EM ICE. Freeze substitution will be performed on a Leica AFS2, and a User might specify a substitution protocol of preference, which will be utilized if compatible with Facility practices. Similarly, for chemical fixation, the User is allowed to specify their protocol of preference, which will be adopted if compatible with Facility practices. For resin-embedding, the User could provide a resin of preference, or alternatively, the currently available resin be used. Sections of resin-embedded sample will be prepared on Leica UC7. A maximum of 1 resin-embedded sample can be processed for unit of service, to be sectioned in ribbons, and to be applied to a maximum of 4 TEM support grids. User might specify thickness of final sections, and these recommendations will be followed if compatible with the Facility practices. User might provide TEM grid support of preference, otherwise the currently available grid support will be used. Imaging will be performed an a Thermo Scientific Spectra 300 kV either in TEM, equipped with CETA 2 camera, or STEM mode in BF\DF\HAADF. Imaging conditions, if requested by the Usermust be compatible with Facility practices. Microscope access is granted for a maximum of 5 days per unit of service, including all steps from sample loading to alignments and collection, and according to Facility Staff availability. All steps of this service will only be performed by Facility Staff. To maximise efficient use and access to high-end TEM instruments, priority will be given to applicants specifically requesting parts of this service (e.g. only sectioning, or only S\TEM imaging of already prepared sections).

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The Visium Spatial Gene Expression solution by 10X Genomics enables spatial profiling of gene expression in intact tissue sections, preserving cellular spatial context. For fresh-frozen tissues, the protocol involves sectioning, mounting, fixation, permeabilization, reverse transcription, library preparation, sequencing, and spatial mapping. For FFPE tissues, additional steps include deparaffinization, rehydration, antigen retrieval, and tissue mounting with Visium Cytassist. Both protocols end with library preparation, sequencing, data analysis, and spatial mapping to retain spatial information.

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Total RNA sequencing comprehensively analyzes the transcriptome, revealing mRNA and non-coding RNA profiles. This technique aids in understanding gene expression, identifying novel transcripts, and deciphering regulatory mechanisms in various biological processes. The process involves RNA extraction, ribosomal RNA depletion, cDNA library generation via reverse transcription, fragmentation, adaptor ligation, PCR amplification, high-throughput sequencing, and bioinformatic analysis for gene expression quantification and transcript discovery.

Libraries are prepared using the Illumina Total RNA Prep protocol (Illumina) with Ribo-Zero Plus that supports diverse RNA inputs, including FFPE and low-quality samples, and incorporates Ribo-Zero Plus or Ribo-Zero Plus Microbiome for efficient removal of abundant RNA from various species, including human, mouse, rat, bacteria, and complex microbial samples like stool, ideal for meta-transcriptomic studies.

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Our service focuses on introducing INDELs (frame-shift mutations) to generate gene knock-out lines (KO), each targeting a single gene in either hPSCs or immortalized/cancer cell lines.

Characterization of each engineered cell line includes:

• Cell identity confirmation using STR analysis
• Confirmation of desired editing via Sanger sequencing.

• Karyotyping (Q-banding) and identification of Copy Number Variations (CNVs) at high resolution
• Master bank post-thaw viability and mycoplasma testing.
• Optional evaluation of undifferentiated stem cell markers and pluripotency markers upon 3-germ layer differentiation assay.

We accept cell lines in a cryopreserved state, with a minimum of 1 x 10^6 cells per cryovial.

We generate up to 3 homozygous knockout (KO) clones. Heterozygous KO clones can be provided upon agreement with the applicant. If technically feasible, clones will be analyzed for the occurrence of allelic dropout.

The service typically requires 2 to 3 months for completion and includes the delivery of 1-3 clones, each provided in 10 cryopreserved vials.

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Small RNA sequencing is crucial for studying short RNA molecules (18-30 nt) involved in gene regulation. It analyzes miRNAs and small RNAs, shedding light on gene regulation, development, and disease. The process involves RNA extraction, size selection, adapter ligation, library amplification, size selection, sequencing, and bioinformatics analysis to identify and quantify small RNAs. High-throughput sequencing reveals small RNA profiles, which are analyzed through bioinformatics to identify miRNAs, siRNAs, and piRNAs, aiding in understanding their roles in cellular processes.

Libraries are prepared using the SMARTer smRNA-Seq Kit (Takara) that is compatible with total RNA or enriched small RNA, incorporating SMART technology and primers with locked nucleic acids (LNAs). Users can analyze various small RNA species and create complex sequencing libraries. Illumina adapters and index sequences are added during library amplification, ensuring unbiased representation of diverse small RNA species.

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Single-cell multiome ATAC + Gene Expression technology by 10X Genomics enables concurrent profiling of chromatin accessibility and gene expression at the single-cell level, offering a holistic view of cellular molecular landscapes. It aids in understanding cellular diversity, identifying cell types, deciphering regulatory networks, and correlating chromatin accessibility with gene expression.

The ATAC-seq component assesses the accessibility of chromatin, offering insights into regions of the genome that are open and accessible for transcription factors and other regulatory elements. Concurrently, the RNA-seq component captures messenger RNA transcripts present in each nucleus, shedding light on active genes and their expression levels.

The workflow involves cell isolation, nuclei preparation, transposase reaction for ATAC-seq, GEM formation, reverse transcription for RNA-seq, library preparation, high-throughput sequencing, and specialized bioinformatics analysis for data processing.

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Single-cell immune profiling with 10x Genomics technology enables 5′ RNA sequencing at the single-cell level, alongside profiling T-cell and/or B-cell receptors at single-cell resolution by sequencing V(D)J regions. Cells are isolated into droplets, where RNA is barcoded at the 5′ end. cDNA synthesis and targeted amplification yields three types of libraries: GEX libraries for single-cell gene expression analysis, and libraries for TCR and BCR gene profiling. Pooled libraries undergo high-throughput Illumina sequencing. Bioinformatics tools demultiplex reads, assign to cells based on barcodes, and identify T and B cell clones, providing insights into gene expression and immune cell repertoire diversity and clonal expansion.

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Single-cell ATAC sequencing with 10X Genomics profiles chromatin accessibility of individual cells/nuclei, revealing cellular heterogeneity and regulatory processes. The scATAC-seq protocol involves cell isolation, nuclei extraction, transposase reaction for chromatin fragmentation and adapter addition, GEM formation for nuclei encapsulation with unique barcodes, library preparation via PCR amplification, high-throughput sequencing, and data analysis to identify accessible chromatin regions.

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Single-cell 3′ RNA sequencing with 10x Genomics technology allows gene expression study at a single-cell level, profiling thousands to tens of thousands of cells in parallel. It captures cellular heterogeneity, identifies rare cell types, and discerns gene expression differences, enhancing understanding of cellular diversity. The process involves cell isolation into droplets, RNA capture and barcoding and cDNA synthesis, library preparation, pooling, sequencing, and bioinformatic analysis for single-cell transcriptomic profiling.

The Single Cell Gene Expression Flex assay by 10X Genomics, enables single-cell/nuclei RNA-seq libraries from formaldehyde-fixed cells and tissues, including FFPE blocks. Utilizing sequence-specific probe pairs for about 18,000 human and 19,000 mouse genes, this method accounts for RNA degradation. Sample multiplexing, facilitated by sample barcodes in probes, allows pooling of discrete cell populations, accommodating up to 16 samples and potentially analyzing 128,000 cells per GEM reaction.

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The NF for Genomics will provide sequencing of pools of libraries prepared by the users with the NovaSeq6000 sequencing platform (Illumina).

The NovaSeq 6000 sequencing platform by Illumina offers high-throughput capabilities, making it ideal for diverse genomic studies. With its scalability and sequencing-by-synthesis technology, it efficiently generates large amounts of sequencing data. Its dual-flow cell design allows parallel sequencing of multiple samples, reducing turnaround times. Common applications include whole genome sequencing, exome sequencing, transcriptome analysis (bulk and single-cell RNA-Seq), metagenomics, epigenomics, and population genomics. This platform is pivotal for comprehensive genomic research, from understanding genetic variations to characterizing microbial communities and studying epigenetic modifications.

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The NF for Genomics will provide sequencing of pools of libraries prepared by the users with the NextSeq2000 sequencing platform (Illumina).

The NextSeq 2000, an Illumina platform, is renowned for its high-throughput benchtop sequencing capabilities, offering flexibility and scalability for diverse applications. It employs sequencing-by-synthesis technology, capturing emitted fluorescence to determine DNA sequences. With versatile configurations, it supports various flow cell setups and sequencing kits, catering to different project scales. Commonly used for whole genome sequencing (WGS), RNA sequencing (RNA-Seq), targeted sequencing (including amplicon sequencing and target capture), exome sequencing, metagenomics, and ChIP-Seq and epigenomics studies, the NextSeq 2000 is pivotal in genomics research, enabling comprehensive analysis of genetic and epigenetic features across diverse biological samples.

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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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Direct RNA Sequencing with Nanopore technology sequences RNA molecules without cDNA conversion, offering real-time detection of RNA sequences and modifications. It preserves RNA’s native state, revealing insights into RNA processing and modifications. RNA extraction, adapter ligation, sequencing, and data analysis are key steps. Raw signals are base-called for RNA sequence reconstruction. This method can offer a comprehensive view of the transcriptome.

The Direct RNA Sequencing Kit (SQK-RNA004) facilitates native RNA sequencing, avoiding cDNA conversion. It supports poly(A)-tailed RNA or total RNA like eukaryotic mRNA and viral RNA. This upgrade enhances sequencing output and accuracy on the latest RNA flow cells (FLO-MIN004RA and FLO-PRO004RA). It includes reformulated priming reagents for flow cell compatibility and features fuel fix technology for extended experiment runs without additional fuel.

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Cell-free DNA (cfDNA) sequencing with Nanopore technology allows direct interrogation of genetic information in circulating DNA without PCR amplification. This real-time protocol enables methylation status analysis. It offers insights into cfDNA’s genomic landscape, benefiting clinical diagnostics like cancer detection, treatment monitoring, and minimal residual disease identification. The protocol involves cfDNA extraction, library preparation, loading onto a Nanopore sequencing device, real-time sequencing, and data analysis for variant calling, copy number and methylation status analysis.

The library preparation involves repairing DNA ends, dA-tailing, and ligating sequencing adapters. The kit ensures high sequencing accuracies (Q20+) on nanopore Flowcells R10.4.1, with updates for enhanced DNA capture and fuel fix technology for longer runs. The protocol is optimized for short DNA fragments recovery, based on a modified long-reads protocol.

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Nanopore cDNA sequencing allows the exploration of gene expression dynamics, alternative splicing, and RNA biology with long-read capabilities. The process involves RNA extraction, cDNA synthesis, library preparation with unique barcodes, loading onto a nanopore sequencer, real-time sequencing of cDNA strands, base calling, and subsequent bioinformatics analysis for aligning reads, identifying gene isoforms, and quantifying gene expression.

The libraries are prepared with the PCR-cDNA Sequencing Kit that enables nanopore sequencing of cDNA from low input poly(A)+ RNA or total RNA with additional optimization. It employs a strand-switching method to select full-length transcripts and incorporates unique molecular identifiers (UMIs). The kit includes the Rapid Adapter T (RAP T) for enhanced capture and fuel fix technology for longer experiments without fuel addition. A new cDNA RT adapter and RT primer reduce overlaps during reverse transcription and allow measurement of polyA+ tail lengths.

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mRNA sequencing analyzes the transcriptome, revealing gene expression patterns and novel transcripts in cells or tissues. The process involves RNA isolation, cDNA synthesis via reverse transcription, library preparation with added adapters, high-throughput sequencing, and bioinformatic analysis mapping reads to a reference genome or transcriptome to discern gene expression levels and novel transcripts.

mRNA sequencing from standard RNA input is performed with Illumina Stranded mRNA Prep protocol (Illumina) that ensures precise strand orientation measurement, uniform coverage, and high-confidence detection of novel features like isoforms and gene fusions.

mRNA sequencing from RNA low input is performed with the SMART-Seq v4 PLUS Kit (Takara). SMART technology ensures full-length transcript information, enabling analysis of isoforms, gene fusions, and mutations, with improved gene detection via locked nucleic acid (LNA) technology. High reproducibility and accurate coverage of GC-rich transcripts are ensured.

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The Biophysics Unit is equipped with instruments designed to determine the strength and biophysical properties of macromolecular or protein-ligand interactions, including association and dissociation constants, as well as stoichiometry of binding. Available techniques include isothermal titration calorimetry, microscale thermophoresis, and bio-layer interferometry (which can also be utilized for quantifying known components in complex mixtures).

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The services we provide include, but are not necessarily limited to, the following use-cases:

• • Image restoration and denoising: Removing pixel-independent noise from images to increase SNR.
  • Semantic and Instance segmentation: Identifying and segmenting objects in an image, generating image masks.
  • Quantitative Image Analysis: Quantification of intensity levels in images or segmented objects.
  • Morphometric Analysis: Analysis of shape and morphology of segmented objects
  • Custom pipeline development: Construction of an analysis pipeline combining two or more individual steps.

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Motorized, inverted wide-field microscope with digital clearing. Epifluorecence illuminator with 8 single LEDs (395 nm, 438 nm, 475 nm, 551 nm, 555 nm, 575 nm, 635 nm and 730 nm) and sCMOS camera.

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Confocal microscope with STED module and tandem scanners (conventional and resonant), white laser (up to 8 simultaneous laser lines between 440 nm and 790nm), 405 nm laser, AOBS tunable dichroic and integrated FLIM module.

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Point-scanning confocal microscope with white light laser (up to 8 simultaneous laser lines between 440 nm and 790nm), 405 nm laser, AOBS tunable dichroic, dual scanner (conventional and resonant) and integrated FLIM module.

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If the cell is supplied with a donor DNA template, Cas9-induced DSBs can be repaired by integrating DNA sequences of various lengths using homology directed repair (HDR) mechanism. We provide Knock-In (KI) services to obtain reporter cell lines (including safe harbor integrations), edited cells harboring specific single nucleotide corrections as well as labelled proteins (e.g., with fluorescence or a protein tag).

Characterization of each engineered cell line includes:

• Cell identity confirmation using STR analysis
• Confirmation of desired editing via Sanger sequencing.

• Karyotyping (Q-banding) and identification of Copy Number Variations (CNVs) at high resolution
• Master bank post-thaw viability and mycoplasma testing.
• Optional evaluation of undifferentiated stem cell markers and pluripotency markers upon 3-germ layer differentiation assay.

We accept cell lines in a cryopreserved state, with a minimum of 1 x 10^6 cells per cryovial.

The service typically requires 2 to 4 months for completion and includes the delivery of 1-3 clones, each provided in 10 cryopreserved vials.

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MEA assay

MEA arrays measure the electrical activity of large cellular networks in real time without labelling.

• 3BRAIN AG BioCAM DupleX: MEA array system for in vitro functional imaging, compatible with MEA chips with 4096 electrodes. The system works with adherent cells, tissue and organoids slices and allows for recording extracellular voltage oscillations.

Ion Imaging assay

Using a fluorescent ion sensor, it is possible to analyze the electrical activity of a cell by measuring the intracellular ion oscillations using a conventional microscope. This service will be performed using microscopes of the IU1 Imaging.

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The Structural Proteomics unit will perform integrative structural modelling combining medium\low resolution (from 7 to 30 Å) cryo-EM densities and crosslinking MS data acquired at our Facility. In case of very large systems, negative stain data may also be used. This does not refer to model building in cryo-EM densities, but to calculations of localization of subunits or of areas not observed in EM maps. This task can be performed on data from services SB-IU4-A or SB-IU4-B only. Example software: DisVis, integrative modelling platform (IMP), AlphaLink\AlphaLink2. This service will be performed by Facility Staff, but training will be available if requested, provided basic bioinformatics skills of the User (bash terminal, python).

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This service provides high-resolution cryo-TEM data collection of vitrified specimens. According to instrument availability and experimental needs, data collection will be carried out either on 200kV or 300kV microscope systems. A maximum of 1 specimen can be imaged per unit of service. To ensure efficient usage of high-end microscope time, a maximum of 4 grids can be loaded per unit of service. The User can provide cryo-TEM grids either unmounted or mounted on a Thermo Scientific cartridge. In case of an unmounted grid, clipping of the specimen in Thermo Scientific cartridges will be performed by Facility Staff. In case of User-provided already-clipped grids, these will be inspected by Facility Staff prior to acceptance. Imaging at 200 kV will be performed on a Thermo Scientific Glacios while imaging at 300 kV will be performed on a Thermo Scientific Titan Krios G4, both equipped with a Falcon 4i direct electron detector and a Selectris X energy filter. Imaging conditions (i.e., dose, pixel size, magnification, etc.), if requested by the User, must be compatible with the Facility best practices. Microscope access is granted for a maximum of 24 continuous hours per unit of service, including all steps from clipping to loading and TEM alignments and according to Facility Staff availability. For single-particle acquisition, User might opt for beam-image shift assisted data collection (~ 450 – 600 movies\hour) or stage movement for each hole (~ 100 – 250 movies\hour). This service will only be performed by Facility Staff.

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Full-service sorting of rare populations from heterogeneous samples, cell cloning (single cell deposition into multi-well plates), particle enrichment, and high purity bulk sorts.

• High-recovery and indexed single-cell sorting for sequencing.
• Cell sorting of many cell types including immune cell and hematopoietic stem cell subsets, mesenchymal stem cells, viable cytokine producing cells and general cell sorting approaches for cell lines and transfected cells.

Sorter technical features:

• BD FACSDiscover S8
  This instrument is the latest technological advance, representing a breakthrough solution to combine capabilities of cell separation with multiparametric spectral parameter-based analysis and imaging.  Image based cell selection combined with fluorescently labelled tags for determination of tag local. Sorting of up to 6 populations simultaneously with recovery in numerous devices including 384 well microplates. The sorter is equipped with 5 lasers (349 nm, 405 nm, 488 nm, 561 nm, and 637 nm).
• MoFlo Astrios EQ
  The sorter features dual forward scatter for enhanced detection of small particles such as MV’s and capable of very high-speed bulk sorting. Sorting of up to 6 populations simultaneously with recovery in numerous devices including 1536 well microplates. The sorter is equipped with 6 lasers (355 nm, 405 nm, 488 nm, 561 nm, 592 nm, and 640 nm).

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Analysis of cell populations in suspension using conventional, spectral and imaging analyzers.

Analyzer technical features:

• Cytek Aurora Spectral Analyzer
  The analyzer is designed for high-complexity applications, enabling the use of a wide array of new fluorochrome combinations and capable of resolving challenging dye combinations as well as extracting autofluorescence to enhance resolution of dim markers.  The analyzer is equipped with 5 lasers (355 nm, 405 nm, 488 nm, 561 nm, and 640 nm), spectral detection of 64 channels and is equipped with automated sample loader.
• ImageStream Mark II Imaging Flow Cytometer
  This instrument combines the speed, sensitivity, and phenotyping capabilities of flow cytometry with the detailed imagery and functional insights of microscopy. The analyzer is equipped with 6 lasers (375 nm, 405 nm, 488 nm, 561 nm, 592 nm, and 642 nm) and a 785 nm side scatter laser, a 12-channel spectral detector, and multi-channel brightfield.
• CytoFLEX LX Flow Cytometer
  High speed and multi-parametric analysis for immunophenotyping and micro-vesicle analysis with 6 lasers and 21 fluorescence APD detectors. Laser wavelengths include 355, 405, 488, 561, 640, 808 nm. Sample acquisition may be performed in single tubes or in 96-well plates (flat, V or U bottom). Deep-Well plates may also be used for samples requiring lager volumes. In addition to the standard Side Scatter parameter on the 488 nm laser line, an added side-scatter parameter is configured for the 405 nm laser line.

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This service allows exploring the potential of single molecule techniques. It is dedicated to Users with no prior experience with dynamic single molecule (DSM) approaches who would like to obtain ‘proof of concept’ data necessary to plan future experiments. The unit is equipped with two state-of-the-art instruments: (1) c-trap and (2) c-trap edge. The c-trap relies on confocal scanning, and it is predominantly used for imaging in solution. C-trap edge has an option to switch between wide field fluorescence and TIRF. In addition, c-trap edge allows for label free imaging based on IRM, which makes this instrument ideal for imaging close to the surface. The Unit can assist in a very broad spectrum of experimental approaches.

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This service is dedicated to projects where specific single molecule assays have been already established or, alternatively, should be combined with ‘assay development’ service as a simple ‘package’. Examples of experiments currently performed routinely by the unit: (i) detecting changes to DNA structure upon protein binding e.g., melting, looping, compaction; (ii) characterizing protein behaviour upon binding to DNA (diffusion, direct motion, interactions with other proteins). This service can be performed by the Facility Staff or by a trained User. The training can be provided by the unit as part of this service. Full training typically requires 3-5 days. The User can spend up to 10 days at the Facility. The work can be split into two visits. Original data will be handed to the User as .h5 files. The unit will assist the User in accessing the files using Pylake. In case of kymographs, the Facillty Staff can provide a short training on existing tools such as Lakeview.

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Development and execution of custom gene editing projects.

This service is designed to offer expertise and technical support for the implementation of genome engineering projects that demand customized approaches not presently covered by off-the-shelf knock-out and knock-in editing services.

The proposal should provide a thorough explanation of the rational, goals, and expected outcomes to facilitate a comprehensive evaluation.

Tailored gene editing projects involve completing sequential checkpoints before moving on to the implementation of the proposed experiment (Phase 3). Throughout these phases, significant interaction with the applicant is expected.

• Phase 1 (Strategy design): A tailored strategy will be designed following preliminary meetings and discussed with the applicant before proceeding to the next phase.
• Phase 2 (Strategy validation): The designed approach will undergo testing in the relevant cell line to assess its effectiveness (efficiency, locus accessibility, …).
• Phase 3 (Target editing generation): Upon successful completion of Phase 2, the validated approach will be applied to generate the desired cell line.

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This service provides cryo-Fluorescence Microscopy imaging including sample preparation by plunge-freezing and grids clipping. A maximum of 1 specimen can be processed per unit of service, maximum 4 samples (i.e. replicates) can be prepared per specimen. Glow discharging will be performed by either a Pelco EasyGlow, a Quorum GloQube, or Solarus II plasma cleaner device. Plunge-freezing will be performed on a Thermo Scientific Vitrobot Mk IV or a Leica EM GP2. Applicants may provide TEM grid supports of preference, otherwise the Facility Staff will use the available TEM grid supports. Widefield imaging will be performed on a Leica Thunder cryo-CLEM system. Confocal imaging will be performed on a Leica Stellaris 5 cryo-CLEM system equipped with white light laser. Both microscopes are equipped with a 50x \ 0.9 NA lens. This service is provided for a maximum of 8 continuous hours per unit of service, including plunging, clipping, and imaging. In case of User-provided already-clipped grids (in Thermo Scientific cartridges), these will be inspected by Facility Staff prior to acceptance. This service will only be performed by Facility Staff.

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This service provides sample preparation by plunge-freezing, grid clipping for autoloader system and cryo-TEM imaging at 200 kV. A maximum of 1 specimen and 8 grids can be prepared and processed per unit of service. An optional “polishing” SEC step can be performed on thawed material by the Biophysics Unit, as and if specified in the request for this service. Glow discharging will be performed by either a Pelco EasyGlow, a Quorum GloQube, or Solarus II plasma cleaner device. Plunge-freezing will be performed on a Thermo Scientific Vitrobot Mk IV or a Leica EM GP2. Applicants may provide TEM grid supports of preference, otherwise Facility Staff will use holey TEM grid supports available. Imaging will be provided for a maximum of 8 continuous hours per unit of service. Cryo-TEM imaging will be performed only in EF-TEM mode at 200 kV on a Thermo Scientific Glacios equipped with a Falcon 4i direct electron detector and Selectris X energy filter. Imaging conditions requested by the User, if provided, must be compatible with Facility practices. In case of grids showing optimal particle distribution and ice quality, this service can be extended for overnight data collection, if compatible with Facility Staff working hours. The User can also provide already-made cryo-TEM grids either unmounted or mounted on a Thermo Scientific cartridge. In case of an unmounted grid, clipping of the specimen in Thermo Scientific cartridges will be performed by Facility Staff. In case of User-provided already-clipped grids, these will be inspected by Facility Staff prior to acceptance. This service will only be performed by Facility Staff.

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The Structural Proteomics unit will perform crosslinking MS on a purified protein complex to identify protein-protein interactions and map residue distances. This service does not include preliminary crosslinking reaction optimisation, which should be performed by the User at their own institution (in consultation with our Facility Staff). This service will only be performed by Facility Staff.

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The Structural Proteomics unit will perform crosslinking MS on a purified protein complex to identify protein-protein interactions and residue distances. This service includes preliminary crosslinking reaction optimisation and proteomics acquisitions. These can be performed by our Facility Staff provided the protein sample, or by a visiting User with our assistance.

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The Structural Proteomics unit will perform crosslinking MS to characterize the interactome of a particular target protein in situ. Crosslinking reactions can be performed on cells, in lysate or after competitive or otherwise non-denaturing (native) elution. As this experiment requires a careful and at times tricky design, preliminary results will be shared with the User and guidance provided to set or optimize the experimental conditions. This service will be performed by Facility Staff if the crosslinking reaction occurs at the home institution with guidance from us, or by a visiting User under our direct supervision if the entire workflow is requested.

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The Structural Proteomics unit will perform crosslinking MS to characterize the topology of the protein-protein interactome of a particular target protein or enriched cellular fraction in situ. Examples include: interactome of pulldowns with an endogenously tagged protein of interest; interactome of vesicles\cellular compartments; interactome of bacterial cells or virus\host interactions after pulldown of specific virulence factors. Crosslinking reactions can be performed on cells, on lysates or after competitive or otherwise non-denaturing (native) elution. As these experiments require careful design, the User will be granted access to preliminary acquisitions to characterise the sample and set the correct reaction conditions, or guidance to perform the optimization at their home institution. This service will be performed by our Facility Staff in collaboration with a visiting User.

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Autonomous Flow Cytometry Analysis (after completion of a training session) or Operator Assisted Flow Cytometry Analysis.

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Structural biology and biochemical assays often require sample optimization to achieve stability and homogeneity, while understanding the oligomerisation state and precise composition of macromolecular complexes is crucial for unravelling their architecture. To address these needs, the Biophysics Unit offers a service leveraging various instruments to determine the molecular weights of species in solution and their thermal stability parameters. Techniques utilized include mass photometry, SEC-MALS, dynamic light scattering, and nanoDSF.

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This service is dedicated to projects aiming to explore and develop complex biochemical assays or assays that had never been performed before. Examples: activity assays (e.g., transcription), motility surface assays, cell manipulation assays. This service will only be performed by Facility Staff, ideally accompanied by the User. As part of this service, the Facility Staff will check sample quality, troubleshoot, and define optimal conditions for data acquisition. If possible, an automation protocol for the experiment can be developed to streamline future data collection. The User can spend up to 10 days in the Facility, testing various samples. The work can be split into two visits. The outcome of this service is an established protocol that can be used for ‘data acquisition’ service.

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Microbiome analysis via 16S and ITS amplicon sequencing is pivotal for studying microbial community composition and diversity, spanning bacteria and fungi. The 16S rRNA gene, found in bacteria and archaea, and ITS for fungi are key markers used for taxonomic classification.

The process entails sample collection, DNA extraction, PCR amplification targeting variable regions of 16S rRNA or ITS genes, library preparation, and high-throughput sequencing. Taxonomic classification involves clustering reads into operational taxonomic units (OTUs) or amplicon sequence variants (ASVs), followed by comparison to reference databases. Diversity and community analyses assess microbial community structure using metrics like alpha and beta diversity, unveiling richness, evenness, and composition insights.

Libraries are prepared using the QIAseq 16S/ITS Panels (Qiagen), developed for sequencing 16S rRNA and ITS regions on Illumina platforms and to perform a parallel profiling of bacterial and fungal communities.

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STED microscope with 4 laser sources (405 nm, 485 nm, 561 nm, and 640 nm), depletion laser (755 nm), adaptive optics system, MATRIX detectors and FLIM module

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