10X Genomics Single Cell Sequencing
We will be transitioning to the new 10X ‘Next GEM Technology’ for both single cell 3′ GEX and ATAC-seq assays as of April 2020. You can read more about this here. According to 10X, “the Chromium Next GEM Single Cell 3’ Reagent Kits v3.1 and Next GEM Chip G Single Cell Kits provide the same features and comparable assay performance as Single Cell 3’ Reagent Kits v3 and Chip B Single Cell Kits”.
Please schedule any 10X single cell experiment at least a week in advance. We highly advise a consultation prior to experiment scheduling.
We now allow UC Davis users with BSL1 samples to access to our 10X Chromium controller as part of our shared instruments for customer use. This will allow researchers the flexibility to perform single cell experiments with their own reagents. Please contact 10X for training.
The 10X Genomics Single Cell system is the single-cell expression profiling platform enabling the analysis of large cell numbers at the highest capture efficiency (of up to 65%). The technology allows for high-throughput single cell transcriptomics of many different cell types as well as single-nuclei expression profiling. The flexible workflow encapsulates 500 to 20,000 cells or nuclei per library together with micro-beads into nano-droplets. Each bead is loaded with adapters containing one of 750,000 different barcodes for the single cell RNA-seq library preps. In contrast to other protocols (e.g. Drop-Seq) the 10X controller is capable of loading “all” droplets with micro-beads, enabling single-Poisson distribution loading and thus high capture efficiencies (in contrast to double-Poisson loading of other protocols). 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. The resulting data can be analyzed with the free Cell Ranger and Loupe Cell Browser software. In addition the Bioinformatics Core has developed a custom single-cell data analysis pipeline for 10X data.
- By default, our staff will prepare the single-cell RNA-seq (scRNA-seq) libraries from cell suspension samples submitted by the research labs. Since the samples should be processed as quickly as possible, the experiments need to be planned and scheduled together with our staff.
- In case your lab is planning a large number of single-cell experiments, it might make sense for investigators to get trained to be able to process the cell suspensions on the 10X Genomics Chromium controller.
The principles of the 10X Single-Cell RNA-seq library preparation:
10X Chromium Single Cell Features:
- Fast workflow from cell suspension to 3′-cDNA library.
- Captures 100-10,000+ cells per sample and up to 80,000 cells per run in ~18minutes.
- Recovers up to ~65% of cells (typically 50%).
- Low doublet rate (~0.9% per 1,000 cells).
- Compatible with Illumina NovaSeq6000, HiSeq4000, NextSeq, and MiSeq sequencers.
The 10X Single-Cell libraries are most economically sequenced on the Illumina NovaSeq 6000 with paired-end 100bp reads. (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 50,000 reads per cell should be sequenced (for cell types with complex transcriptomes). For isolated nuclei 25,000 reads each are recommended,
- It is possible to work with cryo-preserved cells and methanol fixed, enabling safe sample shipping and batching.
- The cell size limit is comparatively high. Cells can have a diameter of up to 50 µm.
- In addition to cell suspension samples also nuclei suspensions can be studied, enabling the analyses of brain tissues.
- An add-on kit now allows tagging and pooling of human, mouse, and rat cell samples before 10X library prep potentially reducing the costs significantly. This also allows super-loading of a 10X chip channel (with up to 20,000 cells, not supported by 10X Genomics).
- MULTI-Seq reagents even promise to enable the labeling and pooling of hundreds of cell suspension samples.
10X Chromium Single Cell samples:
- For information regarding sample preparation, please refer to the 10X cell preparation guide.
- See the 3′ GEX User Guide to determine the correct cell numbers and for more information about the workflow.
- For cryopreserved samples please submit at least 100,000 cells per sample.
- We also offer 5′ GEX and Immune Cell Profiling.
- 10X has a new demonstrated protocol for methanol fixation, which is compatible with the 10X system.
- The complete UserGuides are available here (3′ GEX) and here (5′ GEX).
Please inquire with our 10X Genomics specialist Diana Burkart-Waco, PhD, for further details.
10X Chromium Single Cell suspension sample requirements:
- Minimum concentration of 100 cells/ul (700 to 1,200 cells/ul optimal range) in a volume of at least 40ul.
- 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). We will require 10 ul sample for the cell counter.
- Maximum concentration of 1200 cells/ul.
- Single cell suspension should be at least 70% live (Trypan Blue stained) and free of visible debris and doublets.
- Recommend cell suspension buffer is PBS/0.5% BSA. Cell suspension buffers should be free of EDTA and Mg++ as well as free of DNAse to be compatible with single-cell assay. Up to 2% BSA content is OK. Please see 10X cell preparation guide.
- For fresh samples submitting 10-40K cells total is best.
- For frozen samples, 100,00 cells are the minimum, but 1 million are better.
Our Countess II Automated Cell Counter (Life Technologies) can assist you with the preparation and QC of the cell suspension. It uses Trypan Blue staining to provide total cell counts, reports viability, and measures average cell size in as little as 10 seconds. Please see the Countess manual.
The Chromium Single Cell ATAC Solution allows users to profile the regulatory landscape of chromatin in thousands of cells. Nuclei are transposed in a bulk solution and then are partitioned into nanoliter-scale Gel Beads in-emulsion (GEMs). This all
- Profile 100 to 10,000 nuclei per reaction.
- Recovers up to ~65% of cells (typically 50%).
- Tested on cell lines, primary cells, fresh, and cryopreserved samples.
- The complete user guide is available here.
Plate-based scRNA-Seq & Single Cell Genome Sequencing
Individual cells can be sorted by Flow Cytometry Core directly into lysis buffer in 96-well or 384-well plates. This allows for both transcriptome and genome analysis of single cells of a defined highly enrich cell population.
scRNA-seq can be carried out as 3′-end transcript analysis (e.g. using the Qiagen UPX protocol) or as whole transcript analysis (using the SMART-Seq2 protocols) and sequenced on Illumina sequencers. Even full-length transcript sequencing on both PacBio and Oxford Nanopore platforms is possible with SMART-seq amplified cDNA samples.
Both Single-Cell-Genomics and Single-Cell-Transcriptomics are enabled by the Fluidigm C1 Single Cell Automation Prep. This is a single cell capture microfluidic technology that processes up to 500 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.