Sequencing and Single Cell Analysis
1) 10X Single Cell Sequencing Service
Chromium Single Cell Gene Expression provides mRNA profiling at single cell resolution. It provides a comprehensive way to profile gene expression, either whole transcriptome or targeted, alongside Cell Surface Protein or CRISPR edits for multidimensional insights into complex biology. The data can be utilized to 1) analyze how cellular heterogeneity contributes to your biological system with transcriptional profiling at single cell resolution across tens of thousands of cells, 2) gain new insights into cell subtypes and states with multiomic readouts of gene and cell surface protein expression, 3) explore cellular phenotypes with whole transcriptome analysis or focus on relevant genes and pathways using pre-designed or custom Targeted Gene Expression panels and 4) investigate complex genetic networks and perturbed transcriptomes in normal and diseased cell types by simultaneously detecting CRISPR guides and single cell gene expression profiles. The workflow begins with a suspension of single cells or nuclei. For gene expression analysis alone, unlabeled cells or nuclei can be used. Find out more here.
Sample Preparation:
Chromium Single Cell Gene Expression is compatible with fresh, fixed (methanol), and cryopreserved single cell or single nuclei suspensions. Species can range from human, mouse, rat, model organisms, and plants. Sample preparation should be vigorously optimized and tested for each sample type ahead of time.
Resources:
Single Cell Protocols - Cell Preparation Guide
Single Cell Gene Expression Demonstrated Protocol Compatibility Table
Sequencing Requirements:
Illumina® NovaSeq: 100 Cycles
Illumina® NextSeq 1000/2000: 100 Cycles
Recommended Sequencing: Minimum 20,000 read pairs/cell*
Single Cell 3' v3.1 Dual Index Gene Expression
| Read | Read 1 | i7 Index | i5 Index | Read 2 |
| Purpose | Cell barcode/UMI | Sample Index | Sample Index | Transcripts |
| Length** | 28 | 10 | 10 | 90 |
Single Cell 3' v3.1 Single Index Gene Expression
| Read | Read 1 | i7 Index | i5 Index | Read 2 |
| Purpose | Cell barcode/UMI | Sample Index | Sample Index | Transcripts |
| Length** | 28 | 10 | 10 | 90 |
*Adjust sequencing depth for the required performance or application. The Sequencing Saturation metric and curve in the Cell Ranger run summary can be used to optimize sequencing depth for specific sample types (note: this metric was named cDNA PCR Duplication in Cell Ranger 1.1 or earlier).
**Shorter transcript reads may lead to reduced transcriptome alignment rates. Find out more here.
Sample Submission Guideline:
-
- Please fill out sample submission form (please request) and send it to samplesubmission@salk.edu.
- Frozen cells OR nuclei suspension in duplicate can be submitted. Please optimize/test ahead of time to ensure that sample viability reaches > 90% post-thaw. Please remove debris/dead cells before freezing samples.
- Recommended Quantity 1x10^6 Cells or Nuclei in 500µl of appropriate cryopreservation media. (Minimum Quantity: 50,000 cells in 100 µL).
- Shipment Method: Dry Ice overnight.
Description:
The Chromium Single Cell ATAC (Assay for Transposase Accessible Chromatin) solution provides a robust and scalable approach to map the epigenetic landscape at single cell resolution. Using a transposase enzyme to preferentially tag accessible DNA regions with sequencing adaptors, it provides a comprehensive analysis of open chromatin regions to 1) resolve cell types and states using genome-wide, epigenetic profiles with single cell resolution 2) discover cis-regulatory elements that drive gene expression differences between cell types and states 3) characterize cell-specific gene regulatory networks to understand the epigenetic underpinnings of disease, developmental plasticity, and cell identity and 4) identify transcription factors (TFs) that determine cell identity, and perform lineage and developmental tracing.
Find out more here.
Sample Preparation:
A nuclei suspension is needed. It is possible to obtain high quality nuclei suspensions from fresh and cryopreserved cells, fresh tissue, and frozen tissue. This assay has been optimized for human and mouse samples, although other species may be possible.
Resources:
https://support.10xgenomics.com/single-cell-atac/sample-prep
https://kb.10xgenomics.com/hc/en-us/categories/360001072491-Single-Cell-ATAC
Sequencing Requirements:
Illumina® NovaSeq: 100 Cycles
Illumina® NextSeq 1000/2000: 100 Cycles
Recommended Sequencing Depth: 25,000 read pairs per nucleus
Dual-Indexed Sequencing Run: Single Cell ATAC libraries are dual-indexed.
PhiX Spike-In Recommendations: 1%.
Single Cell 3' v3.1 Dual Index Gene Expression
| Read | Read 1 | i7 Index | i5 Index | Read 2 |
| Purpose | Transposed DNA | Sample Index | 10x Barcode | Transposed DNA |
| Length** | 50 | 8 | 16 | 50** |
* Do not pool Chromium Single Cell ATAC libraries with other 10x libraries while sequencing.
** Can be 1 bp shorter to accommodate NovaSeq v1.0 100 cycle kits.
Find out more here.
Sample Submission Guideline:
- Please fill out sample submission form (please request) and send it to samplesubmission@salk.edu.
*** In general, it is highly recommended to proceed immediately to 10X assays upon nuclei isolation without freezing. Thus, it is critical to optimize/test your protocol to freeze/thaw the samples to ensure that the integrity of nuclei remains intact post-thaw. i.e., sample “viability” must reach > 90% post-thaw.
Please remove debris/dead cells before freezing samples. - Only frozen nuclei suspension in duplicate can be submitted. Recommended Quantity 1x10^6Nuclei in 500µl of appropriate cryopreservation media. (Minimum Quantity: 100,000 Nuclei in 100 µL).
- Shipment Method: Dry Ice overnight.
Description:
It enables simultaneous profiling of the transcriptome (using 3’ gene expression) and epigenome (using ATAC-seq) from single cells to deepen your understanding of how genes are expressed and regulated across different cell types.
Find out more here.
Sample Preparation:
A nuclei suspension is needed. It is possible to obtain high quality nuclei suspensions from fresh and cryopreserved cells, fresh tissue, and frozen tissue This assay has been optimized for human and mouse samples.
Resources:
https://support.10xgenomics.com/single-cell-multiome-atac-gex/sample-prep
https://kb.10xgenomics.com/hc/en-us/categories/360004142131-Single-Cell-Multiome-ATAC-Gene-Expression
Sequencing Requirements:
Illumina® NovaSeq: 100 Cycles
Illumina® NextSeq 1000/2000: 100 Cycles
Recommended Sequencing: Minimum 20,000 read pairs/cell*
Single Cell Multiome Gene Expression libraries (Dual Index)
| Read | Read 1 | i7 Index | i5 Index | Read 2 |
| Purpose | Cell barcode/UMI | Sample Index | Sample Index | Transcripts |
| Length** | 28 | 10 | 10 | 90 |
Single Cell Multiome ATAC Libraries
| Read | Read 1 | i7 Index | i5 Index | Read 2 |
| Purpose | Cell barcode/UMI | Sample Index | Sample Index | Transcripts |
| Length** | 50** | 8 | 24 | 49 |
*Adjust sequencing depth for the required performance or application. The Sequencing Saturation metric and curve in the Cell Ranger ARC run summary can be used to optimize sequencing depth for specific sample types.
** Sequence length can be adjusted based on sequencing kit used.
Find out more here.
Sample Submission Guideline:
- Please fill out sample submission form (please request) and send it to samplesubmission@salk.edu.
*** In general, it is highly recommended to proceed immediately to 10X assays upon nuclei isolation without freezing. Thus, it is critical to optimize/test your protocol to freeze/thaw the samples to ensure that the integrity of nuclei remains intact post-thaw. i.e., sample “viability” must reach > 90% post-thaw. - Only frozen nuclei suspension in duplicate can be submitted. Recommended Quantity 1x10^6Nuclei in 500µl of appropriate cryopreservation media. (Minimum Quantity: 100,000 Nuclei in 100 µL).
- Shipment Method: Dry Ice overnight.
2) Single Cell SMART-Seq® v4 Service
Description:
The SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing is designed to generate high-quality, full-length cDNA directly from 1–1,000 cells or 10 pg–10 ng of total RNA, in a convenient input volume of 1–10 µl. This application provides the benefit of generating cDNA using proprietary SMART® (Switching Mechanism at 5ʹ End of RNA Template) technology. This technology relies on the template switching activity of reverse transcriptases to enrich for full-length cDNAs and to add defined PCR adapters directly to both ends of the first-strand cDNA. This ensures the final cDNA libraries contain the 5ʹ end of the mRNA and maintain a true representation of the original mRNA transcripts; these factors are critical for transcriptome sequencing and gene expression analysis. SMART technology offers unparalleled sensitivity and unbiased amplification of cDNA transcripts, and allows direct cDNA synthesis from intact cells.
- Starting Material: Total RNAThis protocol has been optimized for cDNA synthesis starting from 10 pg of total RNA. However, if your RNA sample is not limiting, we recommend that you start with more total RNA. Purified total RNA (10pg- 10ng) should be in 10µl nuclease-free water.
- Starting Material: Cells
- This protocol has been validated to generate cDNA starting from intact cells; it is possible to use this protocol with previously frozen cells. The cDNA synthesis protocol has been tested with cultured cells. It cannot be used with cells that have undergone fixation.
- Prior to lysis, cell suspension should be washed in Mg2+ and Ca2+-free 1X PBS. The presence of media can interfere with the first-strand synthesis.
- After washing, cells can be resuspended directly in 1X Reaction Buffer (provided by SMART-Seq v4) or 1X mild hypotonic lysis buffer [0.2% (vol/vol) Triton X-100 and 2 U/µl RNase inhibitor]. The final volume should not exceed 10.5µl. If using FACS, cells can be sorted directly into 1X Reaction Buffer, 1X mild hypotonic lysis buffer or up to 5 µl of Mg2+ and Ca2+-free 1X PBS.
Sequencing Requirements:
Illumina® NovaSeq: Singe/Paired-End options (SE100, PE50, PE100)
Illumina® NextSeq 1000/2000: Singe/Paired-End options (SE100, PE50, PE100)
Illumina® HiSeq4000: Single End 50-bp run only available
Recommended Sequencing: Minimum 1 million reads/cell*
| Read | Read 1 | i7 Index | i5 Index | Read 2 |
| Purpose | Cell barcode/UMI | Sample Index | Sample Index | Transcripts |
| Length** | 50/100 | 8 | 8 | 50/100 |
*Adjust sequencing depth for the required performance or application.
Sample Submission Guideline:
- Please fill out sample submission form (please request) and send it to samplesubmission@salk.edu.
- a. Total Ultira Low Input RNA samples (Total 10pg-10ng in 10µl) can be submitted in 8-well PCR strip tubes with individually attached caps (USA Scientific #1402-4700). If you need QC, you will need to prepare additional 5 µl of samples in separate 8-well PCR strip tubes for Qubit OR/AND Agilent Tape Station.b. Cells (single cell) can be sorted directly into a 96 well plate (Thermo Scientific #AB0900) in appropriate buffer and volume, frozen and submitted. No QC will be performed.
- Shipment Method: Dry Ice overnight.
