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

    Coming Soon

  • CITE-Seq Equivilent Method
  • ATAC-Seq
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

Genome

  • High-quality libraries from only 1 ng of genomic DNA
  • Call and phase major classes of structural variants (SVs) like deletions, inversions, and translocations, even in genes inaccessible to short-read sequencers
  • Phase SNVs, indels and SVs across >10 Mb haplotype blocks

Exome

  • High-quality libraries from only 1 ng of genomic DNA
  • Detect large scale structural variants (SVs) including gene fusions
  • Call and phase variants across the entire exome
  • Detect variants in genes inaccessible to standard short-read libraries
  • Useful for high sequence homology, including problematic duplications, inversions and pseudogenes
  • Optimized baits utilize Linked-Reads for intron-spanning

DeNovo Assembly

  • True diploid assembly unlike other methods
  • Fast and simple de novo assembly workflow
  • Go from sample to assembly in <2 weeks
  • 10 Mb scaffold-size and long-range accuracy
  • High-quality assembly from as little as 1 ng of DNA

Current 10X DNA Methods Available

Chromium Genome (& Genome DeNovo Assembly) & Chromium Exome

The Chromium Genome & Exome Protocols generate long-range information across the length of individual DNA molecules. For this solution, starting the process with high molecular weight (HMW) genomic DNA (gDNA) results in better performance, such as increased haplotype phase block length and ability to call structural variants. Optimal performance has been characterized on input gDNA with a mean length greater than 65 kb, and the method outlines the extraction of HMW gDNA with optimal size from live cells. See picture below:

Reads (short blue lines) are generated from each high-molecular weight gDNA molecule (long blue line). Reads from the same molecule will share the same barcode (gold, orange, green, blue, black or purple). Linked-Reads allow increased physical coverage that provides the ability to link distant loci and reconstruct long range haplotypes. For example, linked reads in this example illustrate that the three loci (A, B and C) can be linked. See picture below:

DeNovo Assembly of diploid genomes from 1GB to 3.5Gb can be assembled using linked-read technology, preserving haplotype information in the process.

Genome Library Structure:

Overview of Chromium Genome & Exome Methods:

Genome & Exome Protocol Steps Following Isolation of High Molecular Weight DNA Fragments

Recommended Sequencing Parameters and Sequencing Depth:

Sequencing Read Recommended Number of Cycles for Genome Protocol Recommended Number of Cycles for Exome Protocol
Read 1 150 cycles 100 cycles
i7 Index 8 cycles 8 cycles
i5 Index 0 cycles 0 cycles
Read 2 150 cycles 100 cycles

Specifications Recommended Coverage for Genome Protocol (Human) Recommended Coverage for Exome Protocol (Human)
Gigabases of Sequence 128 9
Reads Passing Filter 850 Million (425 Million Read Pairs) 90 Million (45 Million Read Pairs)
Targeted Duduped Depth >30x >60x

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.