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10x genomics sample preparation protocol

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Isolation of Nuclei for Single Cell RNA Sequencing

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Europe Asia Pacific North America. Analytical Biosciences Limited Beijing, China. Analytical Biosciences was founded to create and harness the human disease precision atlas through cutting-edge single-cell genomics and bioinformatics, provide integrated and efficient solutions to partners in the biological, clinical and pharmaceutical fields. Now, we had developed several fast and efficient single cell omics analysis methods, established the international advanced single cell omics database and interactive single cell data display platform, and owning a number of single cell biological information technology patents.

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BRC-Seq is a Next-Generation Sequencing Core that sets out to assist researchers with generating high-quality data and providing full workflows to investigators with little experience in sample preparation, quality control, or informatic analysis. Located at the Biomedical Research Centre at the University of British Columbia, we strive to make the power of sequencing available to all researchers at UBC and beyond.

CapitalBio Technology Co. CapitalBio Technology is an industry-leading life science company that develops and commercializes total health-care solutions.

Our company provides a comprehensive portfolio of top-quality products to a diverse customer base ranging from biomedical researchers to healthcare professionals. Our products include an innovative and broad spectrum of microarray and microfluidic chips and related instruments, software and databases, reagents and consumables for basic and translational research, drug development, clinical diagnostics, biosafety and food safety, and molecular breeding.

The Genomics Core enables investigators with little experience in sample preparation, quality control or analysis to interrogate the genome. To facilitate this, we have developed general but complete wet-lab and bioinformatics analysis pipelines to accommodate standard needs and enable most genomewide investigations. For more sophisticated or custom designs, we work actively in collaboration with other Cedars-Sinai cores and resources to facilitate investigations.

DNA Link, Inc. Seoul, South Korea. From sample preparation to bioinformatics support, DNA Link provides end-to-end solution for any genomic research projects.

Genergy Bio-technology Shanghai Co. ShanghaiChina. We own an integrated CRO service platform, including single cell research, high-throughput sequencing, and microarray services. Geninus Seoul, South Korea. Geninus, Inc. Their database has both genetic and clinical information to enable clinicians and researchers to promote public health. They lead next generation precision medicine by constructing clinical-genetic information big data.

The core proprietary technology is powered by Samsung Genome Institute and is continuously developing by collaborating with Samsung Medical Center and many renowned research institutes around the world. Icahn School of Medicine at Mt.Cell suspensions containing to 16, cells per sample are loaded into a Chromium Chip B along with partitioning oil, reverse transcription reagents, and a collection of gel beads that contain 3, unique 10X Barcodes.

An emulsion is created as the cells and reagents are passed through the microfluidic channels of the Chip B microfluidic device during a 9-minute run in the 10X Genomics Chromium Controller. Appointments for 10X Genomics experiments can be scheduled with Opal Allen opal. Three time slots 9 am, 11 am, and 1 pm are available on Monday through Thursday of each week. We recommend finalizing the appointment at least two to three weeks in advance to ensure that the desired date and time slot are available for your experiment.

Can I cancel a 10X Genomics appointment due to poor cell viability, low cell concentration, or a failure of cells to achieve the desired growth state? Researchers can cancel a 10X Genomics appointment. Can I perform a practice cell preparation and acquire feedback from the HTG Shared Resource in regards to the quality of the cell suspension and viability of the cells? We strongly encourage researchers to perform a practice cell preparation prior to their first single cell experiment on the 10X Genomics platform.

What learning resources are available for sample preparation on the 10X Genomics website? Instructional videos that describe the library preparation process are also available.

10x genomics sample preparation protocol

The 10X Genomics Cell Preparation Guide describes best practices and methods for washing, concentrating, resuspending, and counting cells.

Cells should be washed a minimum of two times and resuspended in 1X PBS with 0. Resuspension of cells should be performed by slow pipetting using wide bore pipette tips. Chromium microfluidic chips are one-time use consumables that can simultaneously partition up to eight samples per run.

Single cell RNA sequencing experiments routinely target the retention of to 10, cells which require an input of to 16, cells. However, when a high percent of dead cells is present within a cell suspension, an apparent lower cell recovery rate will likely result in the sequencing data. How is single cell resolution achieved with the 10X Genomics single cell platform?

The remaining droplets are largely occupied by a single cell. A low rate of multiplet occupancy will be experienced in single cell experiments and this rate is observed to increase when higher quantities of cells are loaded into the Chip B partitioning device. What is the estimated doublet rate in a 10X Genomics single cell experiment?

The percentage of emulsion droplets containing two or more cells is influenced by the quantity of cells loaded into the Chip B partitioning device and the prevalence of cell aggregates within the cell suspension. Assuming the absence of cell aggregates, the following table, adopted from the 10X Genomics User Guide, can be used to estimate the occurrence of multiplet partitions within an emulsion. Both live and dead cells must be considered when estimating the anticipated doublet rate that will be obtained following partitioning of a cell suspension for a 10X Genomics experiment.

Therefore, a high percentage of dead cells will impact the targeted number of live cells that can be acquired for single cell analysis. A wetting failure is characterized by the absence of a uniform emulsion in an outlet well of a microfluidic chip following cell partitioning.

A wetting failure leads to a loss of proper partitioning of single cells and reagents. Common causes of wetting failures include incorrect priming of reagents in the microfluidic channels, presence of air bubbles in the bottom of wells in the microfluidic chips, presence of surfactant or other viscous reagents in the cell media, or excessive presence of cell aggregates.

We hope researchers will carefully consider cell preparation methods that reduce cell debris and aggregates such as to minimize the occurrence of wetting failures.

Provided that excess sample is available, cell partitioning will be repeated for any sample that fails to form a proper emulsion on the first pass. However, if additional sample is not available, adjacent wells containing other samples that formed a proper emulsion will be processed through library prep as we are unable to replace the reagents used for those samples that successfully formed an emulsion.

Can I recover my sample from the microfluidic Chip B in the event of a wetting failure? Unfortunately, cell suspensions cannot be recovered from microfluidic chips following the event of a wetting failure. How is the 10X Genomics Reverse Transcription primer used to enable the identification of molecules within a library that originate from the same cell?

A total of 3. During cell partitioning, each cell is combined with a single gel bead that is loaded with reverse transcription primers that include one of the 10X Barcode sequences. These primers are used to reverse transcribe mRNA molecules that are released within the partition following cell lysis. The 10X barcode therefore marks the newly synthesized cDNA with a unique 16 nucleotide sequence that is common to all cDNA molecules localized within the same partition.As of January10X Genomics has announced that it is discontinuing production of the 10X Chromium Genome linked read reagents.

We are sad to see this very popular assay go. We have a limited supply in the lab at this time and will continue to offer the assay until our reagent stocks run out likely later in The sequencing libraries are generated with the Chromium controller instrument and reagents from 10X Genomicsfollowed by sequencing on our Illumina HiSeq sequencers.

Please see these slides for details on GemCode technology and some of the possible applications. Please note that the individual DNA molecules are not meant to be fully assembled with this approach. A single HiSeq lane generates sufficient information for phasing and structural variant analysis of a human sized genome. Human phasing can be carried out using both whole genome shotgun sequencing as well as employing exome capture enrichment. Please see this suggested protocol.

The quality of the DNA samples should be verified by pulsed-field gel electrophoresis. For DNA samples significantly longer than 50 kb these pre-treatments will likely not be necessary. The first step of the actual 10X library prep is a denaturation of the sample.

Thus nicks present in the DNA sample will significantly reduce the linked-read length. The 10X Genomics Chromium Genome assays do require paired-end bp read sequencing. Please note that the 10X Genomics Chromium technology was designed primarily for the analysis of human genomes.

Fresh Frozen Human Peripheral Blood Mononuclear Cells for Single Cell RNA Sequencing

Thus, the design is optimized for large genomes and works best for genome sizes around 3 Gb. A bird genome assembly showed a contig N50 of kb and a scaffold N50 of 18 Mb for a 1. The means the assembly metrics are limited by the karyotype comprising multiple micro-chromosomes. This assembly was based on a single 10X library and a single HiSeq lane of data.

Please note that the Supernova genome assembler was designed only for diploid genomes. As in most cases plant genomes tend to be more difficult to assemble. In general, the results are better than other assemblies based on Illumina sequencing data using shotgun and mate-pair data.

Please contact Diana at dburkart ucdavis. Before submitting samples: The DNA quality should best be verified by pulsed field electrophoresis. In the absence of PFGE equipment you could run the sample on a conventional agarose gel 0. Please email us a gel image before shipping the samples. Fluorometry e. Recent Posts. Latest Tweets. Important Information.Find information and resources for current and returning patients.

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The ATGC provides a complete next generation sequencing service. Project consultation and budget planning with a facility representative and a MDACC faculty bioinformatician. It is created by shearing DNA into base fragments. HiSeq - the Illumina Hiseq is a mid-throughput sequencer that consists of two, eight lane flow cells. The flow cells can be run independently or in parallel.

Each flow has a maximum out-put of Gb per run or It has the longest read-length in the Illumina line-up, generating up to bases per bp paired end run. A variety of flow cells and read lengths provide flexibility on this single sample platform. The ATGC offers several options for transcriptome analysis.

Note: Strand specificity -Preserves strand information. Strand specificity can be used to identify antisense transcripts, determine the transcribed strand of non-coding RNAs and may help to demarcate the boundaries of overlapping genes.

We strongly recommend that first time NGS service users and investigators with large-scale projects schedule a meeting before initiating a project. To schedule a consultation meeting please contact Erika Thompson ejthomps mdanderson. Sample Submission All samples should be accompanied by a completed sample submission form.

Sample submission requirements mimunum quantity and recommended sequence length vary based on the service and sample type.

10x genomics sample preparation protocol

The facility quantifies all samples, performs sequencing reactions, cleanup and capillary electrophoresis, analyzes the data and provides sequence as text files and chromatograms. View our service pricing schedule for more information about DNA sequencing pricing. Longer turnaround times may occur when our sample volume is very high, when special conditions are requested and when we experience instrument problems. The DNA and primer must be submitted in 0. All DNA submitted to the facility needs to be accurately quantified.

Alternatively DNA concentration can be determined fluorometrically. DNA concentrations determined using a spectrophotometer are often artificially high due to the presence of RNA, proteins, bacterial genomic DNA and other contaminants.

Note: Low DNA concentration is the most common cause of poor quality sequence and failed reactions. Too much DNA can, however, be as bad as too little. The presence of too much template results in top-heavy data strong peaks at the beginning which fade rapidlypull-up peaks non-specific peaks that appear below the correct peak and loss of peak resolution. In addition, it shortens the life of our capillaries.

The facility will repeat a sample at no cost to the investigator if there is an instrument failure, or if the quality of sequence is compromised due to an error in the facility. If the investigator recommends a sample be repeated, the same DNA and primer will be used to repeat the sequencing reaction.Inside Visium Spatial Technology.

Cancer Research Highlights Flyer. Cancer Research Brochure. Uncovering the layers of immune cell complexity. Inside the Chromium Controller. Chromium Genome Sequencing Solution Brochure. Chromium Exome Sequencing Solution Brochure. Chromium de novo Assembly Solution Brochure.

TotalSeq™-A Antibodies and Cell Hashing with 10x Single Cell 3' Reagent Kit v2

Chromium de novo Assembly Solution Product Sheet. Transcriptional Profiling of 1. Discover Genetic Heterogeneity in Cancer. Characterization of the Tumor Microenvironment.

In-depth analysis of T cell repertoires using a multi-omic single cell approach. High throughput immune profiling using integrated single cell multi-omics analyses. Single cell ATAC-seq for characterization of complex biological systems. Detection of structural variants using linked-reads with novel algorithms. Haplotype resolved analysis and improved genome coverage with Linked-Reads. Simultaneous single cell analysis of multiple analytes resolves T cell populations at high resolution.

Deciphering the tumor microenvironment cell by cell. Dissecting the microenvironment of multiple tumors by transcriptional profiling and immune repertoire sequencing. Single cell transcriptomics for characterization of complex cellular systems. Full spectrum genome analysis with Linked-Read sequencing. How to determine the genome sequence of single insects. Assembly of individual chromosomes at megabase scale using 10x Linked-Reads.

Resolving the full spectrum of human genetic variation using Linked-Reads. Single cell transcriptomics for characterization of complex systems and biomarker detection.

High throughput Linked-Read sequencing for improved small variant calling. Moving into the darkness: Improving variant analysis with Linked-Reads.

Linked-Read sequencing for molecular cytogenetics.Chromium solutions include:. If you have a linked-reads Genome project that you would like completed, please contact us ASAP, so that we can order the kit.

Also, you can contact Dr. Alabady for assistance with grant proposals and for obtaining a letter of support from GGBC. Contact for 10x Genomics Technical Assistance For technical questions about existing or new 10x Genomics projects, please contact the following Staff:. Chromium libraries have a unique structure that requires special sequencing considerations. Each DNA sample is indexed with four i7 barcodes sequenced as a standard i7 index read of 8bp in length. Each i7 barcode is a combination of 8-bases unique barcodes.

The unique barcode used to link reads is found at the beginning of read 1 and is 16bp in length. Please email Kim and Elizabeth at ggbc uga. Be as specific as possible, so that they can more quickly assist you. Table 1. Please inquire for pricing. Table 2. Table 4. Table 6. Click here for more details.

Long Ranger : Generates high-quality haplotype phasing and structural variant calls. If you are are submitting 24 or more samples please submit samples in a well plate and include an Excel file with the sample layout in your order.

Official quotes can be provided upon request. We can process up to 8 samples on a 10X chip at a time. The library prep cost is reflective of that and cost per sample is lowest for 8 samples. Read lengths can be changed as desired, but please note that shorter transcript reads can reduce alignment rates. Generally a minimum of 20, read pairs per cell is needed. The type and number of sequencing run s depends on the number of samples and coverage you need.

We can make libraries with between and 10, cells per sample, though the recommended starting point is 1, cells the first time you do the experiment. It is recommended to try the protocol with just one sample first before moving forward with a larger number. Customers should work with the tissue in their lab and provide us with a prepared cell suspension. Cell preparation methods can vary between different tissue types. Cells are preferable in most cases, but nuclei can be used as well.

When you submit your order and have the cell suspension ready for library prep we need an accurate measure of cell concentration and viability. These QC data are critical to the protocol working correctly. An inaccurate concentration could clog the chip or result in low yield, and the presence of dead cells will reduce the recovery rate and result in a high level of noise in the data. It is also important to minimize the time from cell prep to loading the 10X chip.

We do not have the equipment for cell counting and viability here at GGBC, so we have to rely on your data. There is a flow cytometer in another core lab on campus that you can use.Please schedule any 10X single cell experiment at least a week in advance. We highly advise a consultation prior to experiment scheduling.

This will allow researchers the flexibility to perform single cell experiments with their own reagents. Please contact 10X for training. In contrast to other protocols e. The single-cell encapsulating process is significantly faster compared to inDrop or Drop-Seq. Up to eight samples can be processed per batch within minutes. Please see the Countess manual.

Nuclei are transposed in a bulk solution and then are partitioned into nanoliter-scale Gel Beads in-emulsion GEMs. This all. Individual cells can be sorted by Flow Cytometry Core directly into lysis buffer in well or well plates. This is a single cell capture microfluidic technology that processes up to single cells to extract single cell transcriptome, reverse transcribe, pre-amplify and ultimately detect and analyze cell activity at the single cell level.

Protocols for genomic analysis of single cells are under development. You can read more about this here. Since the samples should be processed as quickly as possible, the experiments need to be planned and scheduled together with our staff. The assay requires at least a 28 cycle forward read, an 8 bp index read, and a 98 cycle reverse read.

For the most applications an average of 50, reads per cell should be sequenced for cell types with complex transcriptomes. The cell size limit is comparatively high.

This also allows super-loading of a 10X chip channel with up to 20, cells, not supported by 10X Genomics. MULTI-Seq reagents even promise to enable the labeling and pooling of hundreds of cell suspension samples.

If at all possible, please provide 70ul of single cell suspension two attempts at chip loading in case of clog plus additional for cell QC.

10x genomics sample preparation protocol

We will require 10 ul sample for the cell counter. This all Profile to 10, nuclei per reaction. Tested on cell lines, primary cells, fresh, and cryopreserved samples. The complete user guide is available here. Recent Posts.

10x genomics sample preparation protocol

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