Coming Soon
- CITE-Seq Equivilent Method
- ATAC-Seq
Single Cell & Other 10X Applications
The 10X Genomics Chromium Controller instrument is designed to rapidly automate the capture of thousands of single cells or DNA fragments, combining them with reagents in emulsion droplets to perform a variety of assays.
While some information is provided here on our site, we strongly recommend you obtain the latest methods, protocols and kit details from the support section of the 10X Genomics website.
Current 10X Single Cell Methods Available
General Single Cell Application Features
- Capture 100 to 80,000 cells in ~10 minutes
- Capture 1 to 8 cell sample pools per instrument run
- Cell capture efficiency ~65%
- Cell doublet-rate ~0.9% per 1,000 cells captured
- Capture efficiency independent of cell size with no lower size limit
- Capture 500 to 80,000 cells in ~10 minutes
- Input cell range needed 800 to 17,400 cells per sample (up to 8 samples simultaneously)
- From cells to cDNA-3'RNA-Seq libraries in one day
- Unique barcode for each cell
- Unique indexes for each cell sample
- UMI's for each transcript
- ~50,000+ sequencing reads per cell recommended
The Single Cell 3' Solution provides a comprehensive, scalable solution for cell characterization and gene expression profiling of hundreds to millions of cells. With a simplified workflow, users can go from cell sample to sequencing library in less than one work day. This assay performs 3' digital gene expression profiling of 500 − 10,000 individual cells per sample. Thousands of nanoliter-scale emulsions are generated, and single cells are captured along with a hydro-gel bead, each containing a unique barcode on its surface (there are 750,000 unique barcodes available). To achieve single cell resolution, the cells are delivered at a limiting dilution, such that the majority (~90- 99%) of generated GEMs contains no cell, while the remainder largely contain a single cell.
Additional reagents; primers containing an Illumina read 1 sequencing primer, a 16 bp 10x Barcode, a 10 bp randomer and a poly-dT primer sequence are mixed with cell lysate and Master Mix. See figure below:
Upon dissolution of the Single Cell 3' Gel Bead in a GEM, primers containing (i) an Illumina R1 sequence (read 1 sequencing primer), (ii) a 16 bp 10x Barcode, (iii) a 10 bp randomer-UMI and (iv) a poly-dT primer sequence are released and mixed with cell lysate and Master Mix. Incubation of the GEMs then produces barcoded, full- length cDNA from poly-adenylated mRNA. After incubation, the GEMs are broken, and the pooled fractions are recovered. Libraries are generated and sequenced from the cDNA and the 10x Barcodes are used to associate individual reads back to the individual partitions. See figure below:
Sequencing parameters for 3'-RNA-Seq Libraries:
50,000 Read pairs per cell:
| Sequencing Read | Recommended Number of Cycles |
|---|---|
| Read 1 | 26 cycles |
| i7 Index | 8 cycles |
| i5 Index | 0 cycles |
| Read 2 | 98 cycles |
This solution offers the option to generate: 1) An enriched library from either T cells or B cells, directly from first-strand cDNA or 2) An enriched T cell library and/or an enriched B cell library, and/or a 5' gene expression library from amplified cDNA from the same cells.
The Single Cell V(D)J Solutions produce V(D)J enriched and 5' gene expression Illumina-ready sequencing libraries. A library comprises standard Illumina paired-end constructs which begin and end with P5 and P7. For V(D)J enriched libraries, Read-1 encodes the 16 bp 10x Barcode, 10 bp UMI, and 13 bp Switch Oligo, as well as the 5' end of an enriched transcript. For 5' gene expression libraries, Read-1 encodes the 16 bp 10x Barcode and 10 bp UMI. Due to Enzymatic Fragmentation, for both libraries Read-2 encodes a random internal fragment of the corresponding insert. Sample index sequences are incorporated as the i7 index read. See picture below:
Sequencing parameters for V(D)J Enriched Libraries:
5,000 Read pairs per cell:
| Sequencing Read | Recommended Number of Cycles |
|---|---|
| Read 1 | 150 cycles |
| i7 Index | 8 cycles |
| i5 Index | 0 cycles |
| Read 2 | 150 cycles |
Sequencing parameters for 5' Gene Expression Libraries:
50,000 Read pairs per cell:
| Sequencing Read | Recommended Number of Cycles |
|---|---|
| Read 1 | 26 cycles |
| i7 Index | 8 cycles |
| i5 Index | 0 cycles |
| Read 2 | 98 cycles |
The 10X Chromium controller instrument is available for use in the BPF by trained users. Simply sign up on our calendar, acquire all your necessary reagents in advance, prepare your cells and execute your experiment.
The BPF does NOT provide full service processing of cells on the 10X Chromium instrument. We provide comprehensive training. To request training please email us at nextgen@genome.med.harvard.edu
The 10X Chromium controller is available for use 24 hours per day, 7 days per week. Once properly trained you will be given access to our 10X calendar to sign up to use the instrument. You will also be given card access to the lab where the 10X instrument is located.
- 10X Chromium Controller
- BIO RAD C1000 Touch Thermal Cycler
Experiments are self service but a variety of cell handling recommendations can be found on the 10X website.
| Vendor | Service | Catalog Number | Cost |
|---|---|---|---|
| BPF | Training session for up to four lab members at a time: | 3872 | $361.69 |
| 10X | 10X Instrument use fee | 3873 | $111.64 |
| 10X/BPF | 10X Magnetic Separator (free rental use if returned to us within three business days): | 3667 | $590.32 |
Please contact us at nextgen@genome.med.harvard.edu if you have questions about pricing information for your specific project.
Please refer to the 10X website for the latest protocols and documents.
The BPF does not provide data analysis services but they may be available through the Harvard Chan Bioinformatics Core. Please contact them for more details. We do provide basic Cell Ranger output for 3'-RNA-Seq libraries only as well as bcl files.