RNA Molecular Encapsulation System Samplix Single Cell Analysis

RNA Molecular Encapsulation System Samplix Single Cell Analysis is a multifunctional and user-friendly instrument used for preparing live mammalian or microbial cells, organelles, DNA, or other biological materials for high-resolution downstream analysis. It can achieve single molecule, cell, organelle, nanoparticle encapsulation, and high-throughput sorting. DNA encapsulation enables the rapid screening of large and complex gene libraries.

High fidelity gene library preparation instrument

RNA Molecular Encapsulation System Samplix Single Cell AnalysisA high-throughput single-cell, single-molecule, nanoparticle emulsion droplet encapsulation and sorting system

wei yiIt can achieve single molecule, cell, organelle, nanoparticle encapsulation, and high-throughput sorting. DNA encapsulation enables the rapid screening of large and complex gene libraries.

Xdroppreparative

RNA Molecular Encapsulation System Samplix Single Cell AnalysisIt is a multifunctional and user-friendly instrument used for preparing live mammalian or microbial cells, organelles, DNA, or other biological materials for high-resolution downstream analysis.

Using our Xdrop kit, Xdrop safely and quickly encapsulates live cells, organelles, or DNA fragments along with assay chemicals in highly stable double emulsion droplets with a skin volume. These droplets are stable through pipetting, vortexing, incubation, flow cytometry, sorting, and even long-term storage. More importantly, cells can be recovered from droplets for expansion.

Along with appropriate analytical techniques, Xdrop applications include:

1. Human immune cells and NK cells

Enzyme secretion of 2 microbial cells

3 mammalian cells secrete cytokines

4. Validate gene editing, including evaluating unexpected on target and off target rearrangements

With the help of Xdrop, researchers can more easily gain a deeper understanding of the cellular functions and genomics of humans, animals, plants, and microorganisms. Xdrop can also generate single lotion droplets for other workflows, including packaging DNA for unbiased genome-wide amplification. We provide the complete set of reagents, kits, and accessories required for Xdrop. In addition, user training is part of our standard Xdrop installation service.

advantage

1 XDrop can wrap living mammalian or microbial cells in highly stable double lotion drops for functional determination, incubation, phenotypic analysis and screening.

2 Xdrop helps answer key questions in engineering cell and gene therapy research.

3 Xdrop supports molecular engineering workflows, including enzyme evolution and pathway engineering.

4 Xdrop has a user-friendly interface and a small footprint, allowing every molecular biology laboratory to use the * functionality of microfluidics.

Double layer emulsifying reagent kit

All reagents are included in the kit, which can encapsulate millions of cells or molecules per operation.

Xdrop SortPreparation and selection of components

This new member of the Xdrop family can generate and sort double lotion droplets in a user-friendly workflow, without any experience in fluorescence based cell sorting.

The workflow starts with capturing 100 kb DNA fragments, small single cells, or other biological materials from highly stable droplets for PCR and enzyme activity evaluation assays. Then, based on the fluorescence of the contents in the droplets, the droplets are sorted to obtain only the samples of interest for high-resolution downstream analysis, such as:

CRISPR editing to verify no hidden unexpected rearrangements in PCR bias

Identify virus and transgenic integration sites

Evaluating enzyme activity at the single-cell level

Perform high-throughput GFP screening

Advantage:

·Being able to selectively enrich long fragments

·High sensitivity - single-molecule detection

·Maintain the intrinsic nature of DNA

·Almost no prior sequence knowledge is required

·Compatible with PacBio, Nanopore, and Illumina sequencing

·Easy setup through automatic microfluidic system

·Shorten the turnaround time from weeks to days

Application:

·Structural changes

·Complex samples (such as cancer biopsies)

·Difficult to sequence regions (such as those rich in GC)

·Genomic gap closure

·Genotyping/Genotyping/Haploid Typing

·Linking resistance genes with hosts

·Sequencing of low abundance regions

·Assessment of repetitive areas

·Samples containing only nanograms of DNA

Supplementary materials

1. DE50 cartridge - used to wrap mammalian cells in double lotion drops

Produce highly stable~100 skin liter double lotion drops to encapsulate mammalian cells and transform large cells into single cells for detection. The workflow of using Xdrop DE50 Cartridge includes cytokine secretion and cell killing assays. Just load the reagents and samples, place the kit into Xdrop, and then run the program.

Simultaneously or individually run 8 samples

Generate up to 500000 droplets per lane within 8 minutes and incubate them with cells in a CO2 incubator

Restore selected cells for expansion

2. DE20 ink cartridge - used to wrap microbial cells, organelles or long DNA fragments in double lotion drops

Generate highly stable double emulsion droplets of approximately 1.5 picoliters using independent Xdrop DE20 columns to encapsulate millions of microbial cells, organelles, or DNA. Each droplet serves as a compartment for single-cell resolution measurement or DNA targeted enrichment. Just load the reagents and samples, place the kit into Xdrop or Xdrop Sort, and then run the program.

Simultaneously or individually run 8 samples

Generate up to 10 million droplets per lane within 45 minutes and incubate them with cells in a CO2 incubator

Restore selected cells for expansion to ensure a pollution-free workflow

3. DE20 sorting box - used for sorting double lotion drops containing DNA or small cells

Use the new Xdrop DE20 sorting box to sort double emulsion droplets containing fluorescent DNA or small cells. This user-friendly system is designed specifically for use with our new instrument Xdrop Sort, without the need for specialized knowledge in fluorescence sorting. Simply load the reagents and samples, place the reagent kit into Xdrop Sort, and then run the sorting program.

Simultaneously run up to 8 samples and sort billions of droplets per day to ensure a pollution-free workflow

4. SE85 ink cartridge - used to wrap DNA, RNA, organelles or small cells in single lotion droplets

Use a separate Xdrop SE85 ink cartridge to generate a single lotion droplet to encapsulate millions of molecules, organelles or cells. Each droplet serves as a compartment for single-molecule or single-cell resolution measurements. Just load the reagents and samples, place the kit into Xdrop or Xdrop Sort, and then run the program.

Simultaneously or individually run 8 samples

Generate over 50000 droplets per lane within 40 seconds to ensure a pollution-free workflow

application

Xdrop uses proprietary microfluidic technology to generate highly stable double emulsion and single emulsion droplets for encapsulating biomaterials.

This multifunctional and user-friendly instrument encapsulates the components of life, such as live mammalian and microbial cells, or long DNA fragments, for a range of downstream purposes, including incubation, analysis, classification, and molecular profiling analysis.

Based on Xdrop's proprietary microfluidic technology, Xdrop Sort has increased the ability of fluorescent separation of double lotion droplets based on packaging biological materials.

Here are some representative applications.

Cellular functional analysis:

·Human immune cells, NK cells

·Enzyme secretion of microbial cells

·Mammalian cells secrete cytokines

Genomics Applications:

·Structural changes

·Complex samples (such as cancer biopsies)

·Difficult to sequence regions (such as those rich in GC)

·Genomic gap closure

·Genotyping/Genotyping/Haploid Typing

·Linking resistance genes with hosts

·Sequencing of low abundance regions

·Assessment of repetitive areas

·Samples containing only nanograms of DNA

Application of Plant Genomics:

·Enriched specific genomic regions in plant varieties

·Revealing the structure of biosynthetic gene clusters in barley varieties without a reference genome

·Fill the gap in the structure of biosynthetic gene clusters in tomato varieties

Application example:

1Accurate amplification of the entire genome

In fact, 99% of target genomes are covered by sequencing reads from libraries more than once.

Using Xdrop dMDA technology to amplify genes can accurately amplify all genes with high fidelity even when the input amount is as low as 1 pg.

2. Gene Phasing

The CYP2D6 * 7 allele contains one single nucleotide polymorphism (SNP), while CYP2D6 * 25A contains 22 SNPs.

Xdrop technology can accurately detect all 23 variants, including Phasing (see figure below).

Xdrop provides support for applications through simple design and indirect sequence capture of long DNA fragments (~100 kb). Combined with long read long sequencing, Xdrop overcomes the difficulty of gene localization in a single genetically heterogeneous sample.

The high fidelity enrichment can detect one or more SNPs in alleles and enable de novo assembly to address structural changes.

3. Monitoring complex structural changes in genes

A detailed analysis of the readings crossing breakpoints revealed the presence of multiple HPV18 integration sites in the region on chromosome 8. The PacBio, Oxford Nanopore, and Illumina datasets support identified integration sites.

Xdrop technology is a new targeted DNA enrichment method, unique in that it can select, enrich, and sequence natural DNA fragments. This allows for enrichment of large genomic regions (approximately 100kb), including unknown areas. Using Xdrop? The long fragments generated by the method are not only suitable for long read sequencing, but can also provide valuable contextual information for short read sequencing.

4. Sequencing of unknown regions of interest (ROI)

PCR and probe based targeted DNA enrichment methods require intensive design optimization and complete region of interest (ROI) knowledge. These traditional methods have also failed in characterizing complex genomic environments (such as repetitive regions, structural variations, GC rich regions) as well as unknown and rearranged regions. To overcome these limitations, Samplix developed a new microfluidic method, the Xdrop enrichment workflow based on indirect sequence capture.

Xdrop can enrich long (~100 kb) target DNA regions that require the design of a single primer pair on the detection sequence, corresponding to a small portion or flanking region of the ROI. This amplicon is specifically designed for detecting, selecting, and enriching full-length ROIs, followed by capture and sequencing.

Using Xdrop? The ability of technology to address these challenging regions has opened the door to solving many other challenging regions, which may represent a large proportion of the genome. Combining Xdrop enrichment with long and short read sequencing techniques can provide the required high resolution to quickly and economically solve complex genomic scenarios, bypassing whole genome sequencing.

5. Identify and confirm CRISPR gene editing operations

Through simple design and indirect sequence capture of long DNA fragments, Xdrop helps identify transgenic insertion sites that are often difficult to find and costly.

The random integration in the construct and the presence of host genome sequences make the identification of integration sites complex. Xdrop makes validating genome engineering simple and clear.

Experimental Procedure

1. Mixed dPCR reagent

Add DNA to the dPCR reaction mixture containing target specific pri mers.

2. On chip droplet production

Distributed on the droplet generator chip to produce water in water (DE) droplets.

3. High resolution droplet polymerase chain reaction

Droplets can run millions of PCR re operations in parallel through thermal cycling. By detecting fluorescent droplets, droplets carrying target DNA molecules can be identified.

4. Counting and separating droplets

Detection and separation are performed using a cell sorting device (FACS), which can collect PCR positive droplets containing target DNA molecules

5. Enrichment of DNA amplification

The enriched single molecule of long DNA fragments is amplified by droplet multi position shift amplification (dMDA) to ensure unbiased DNA amplification and uniform coverage of the target area.

References

1. Population-wide gene disruption in the murine lung epithelium via AAV-mediated delivery of CRISPR-Cas9 components Honglin Chen, Steffen Durinck, Hetal Patel, etc.

Mol. Ther. Methods Clin. Dev. 2022. 27: 431–449; doi: 10.1016/j.omtm.2022.10.016.

2. Target-enriched nanopore sequencing and de novo assembly reveals co-occurrences of complex on-target genomic rearrangements induced by CRISPR-Cas9 in human cells Keyi Geng, Lara G Merino, Linda Wedemann, etc.

Genome Res. 2022. 32(10): 1876–1891; doi: 10.1101/gr.276901.122

3. Characterization of FMR1 repeat expansion and intragenic variants by indirect sequence capture Valentina Grosso, Luca Marcolungo, Simone Maestri,etc.

Front. Genet. 2021. 12: 743230; doi: 10.3389/fgene.2021.743230

4. Generation and analysis of innovative genomically humanized knockin SOD1, TARDBP (TDP-43), and FUS mouse models Anny Devoy, Georgia Price, Francesca De Giorgio,etc.

iScience. 2021. 24(12): 103463; doi: 10.1016/j.isci.2021.103463

5. CRISPR/Cas9 deletions induce adverse on-target genomic effects leading to functional DNA in human cells Keyi Geng, Lara Garcia Merino, Linda Wedemann, etc.

bioRxiv 2021.07.01.450727;

6. Alt-RPL36 downregulates the PI3K-AKT-mTOR signaling pathway by interacting with TMEM24 Xiongwen Cao, Alexandra Khitun, Yang Luo, etc.

Nature Communications 12, 508, 2021. doi: 10.1038/s41467-020-20841-6

7. Reconstruction of the birth of a male sex chromosome present in Atlantic herring Rafati N, Chen J, Herpin A, etc.

Proc Natl Acad Sci U S A. 2020 Sep 29; 117(39):24359-24368. doi: 10.1073/pnas.2009925117. Epub 2020 Sep 16. PMID: 32938798.

8. Verification of CRISPR editing and finding transgenic inserts by Xdrop Indirect sequence capture followed by short- and long- read sequencing Blondal Thorarinn, Gamba Cristina, Jagd Lea M? ller, etc.

Methods. 2021 Jul; 191:68-77. doi: 10.1016/j.ymeth.2021.02.003. Epub 2021 Feb 12. PMID: 33582298.

9. Xdrop: Targeted sequencing of long DNA molecules from low input samples using droplet sorting Madsen EB, H? ijer I, Kvist T, etc.

2020 Sep; 41(9):1671-1679. doi: 10.1002/humu.24063. Epub 2020 Jun 29. PMID: 32516842; PMCID: PMC7496172.

10. Cell-based Long-read whole genome analysis of human single cells Joanna H? rd, Jeff E Mold, Jesper Eisfeldt,etc.

2021bioRxiv 2021.04.13.439527;

11. Corrigendum to 'Generation of a set of isogenic, gene-edited iPSC lines homozygous for all main APOE variants and an APOE knock-out line Schmid B, Prehn KR, Nimsanor N, etc.

Stem Cell Res. 2020 Sep 21; 48:102005. doi: 10.1016/j.scr.2020.102005. Epub ahead of print. Erratum for: Stem Cell Res. 2019 Jan; 34:101349. PMID: 32971461.

Cell therapy, cell therapy, cell killing, immunotherapy, killer cells, cell immunity, mammalian cells, microbial cells, single cells, cell function, gene sequencing, cell genomics, single-cell sequencing, Sanger sequencing, NGS, PCR, T7 endonuclease 1 mismatch detection analysis, tracking insertion deletion TIDE, amplicon analysis (IDAA) for insertion deletion detection, whole genome sequencing WGS, comparative genomic hybridization CGH, Southern blotting Fiber-FISH、FISH、CRISPR/Cas

Samplix Manufacturer Authorization Letter: