Single Cell Analysis

The ATGC offers single cell analysis using the following platforms:

• 10X Genomics Chromium
• Mission Bio Tapestri platforms

Learn more about each of these platforms below.

Submitting samples for 10X single cell processing

Project Consultation - We strongly recommend a consultation meeting before starting your first 10X Genomics project. Consultations are free for investigators utilizing the ATGC's single cell service. To request a meeting please contact David Pollock (dpollock@mdanderson.org) or Erika Thompson (ejthomps@mdanderson.org).

Scheduling Your Experiment - The single cell service is by appointment only. You must have an appointment prior to submitting samples. Submission appointments should be made a minimum of one week in advance of your anticipated submission date. This prevents scheduling conflicts and ensures the appropriate reagents are available and at the correct temperatures for immediate use (having samples sit while we bring reagents to temperature may negatively impact data). We will make an effort to accommodate appointments made 24-48 hours before submission but we cannot guarantee availability.

To schedule a submission appointment, please contact David Pollock (dpollock@mdanderson.org).

Sample Submission – To submit samples please complete a 10X Genomics Single Cell Service Request Form and email the completed form (with an active account number and the appropriate signature) to David Pollock (see email above). For external investigators, an external billing form which includes the purchase order (PO) number and PI signature authorization in addition to an e-copy of the PO are required in addition to the service request form

Single Cell Applications 

3' scRNAseq Gene expression profiling

Applications Highlights

• Gene expression profiling.
• Capture up to 10,000 cells per well of partitioning chip.
• Feature Barcode to allow detection for cell surface proteins or CRISPR editing (see more information below 5' scRNAseq application information).

Workflow - Single cells, RT priming-beads with specific barcode and universal molecular identifier (UMI), as well as lysis and RT reagents are partitioned into oil droplets where the beads are dissolved and cells are lysed for reverse transcription to take place. The droplets are then broken and the pooled single-stranded, barcoded cDNA are amplified and fragmented for library preparation. During the library preparation process, appropriate sequence primer sites and adapters are added so that the final product contains the 10X Barcode, UMI, the appropriately sized cDNA insert and Illumina adapters with P5 and P7 primer sequences for sequencing on an Illumina Sequencer (typically the NovaSeq6000 or NextSeq500).

5’ scRNAseq gene expression with immune profiling

Application highlights

• Gene expression profiling with the added ability to immune profile T cell TCR and B cell Ig for both human and mouse samples.
• Capture up to 10,000 cells per well of partitioning chip.
• Feature Barcode to allow detection of cell surface proteins and/or CRISPR editing (see more information below).

Workflow - The workflow for 5’ gene expression is similar to that of 3’ scRNAseq with the additional ability to enrich for the V(D)J segments expressed in T cells or B cells after cDNA amplification is completed. This provides the capability of running gene expression profiling, TCR profiling, and Ig profiling from the same cell suspension. 

Feature Barcoding for cell surface proteins and CRISPR editing with the 3' and 5' scRNAseq applications.

Both 3’ and 5’ scRNAseq applications also allow for the detection of cell surface proteins and/or CRISPR editing from the same cell by incorporating 10X Genomics’ Feature Barcode technology. Cells stained with an antibody oligo conjugate (AOC) for cell surface protein(s) of interest and/or cells containing CRISPR-mediated perturbations can be detected using this technology.

For the cell surface component, the customer stains their cells with the AOC(s) of interest, wash, and resuspend the cells for sample submission. AOCs that have been tested and successfully used in this protocol come from BioLegend and their Total-Seq antibodies. For 3’ scRNAseq, customers would use AOCs from BioLegend’s Total-Seq B product line. For 5’ scRNAseq, customers would use AOCs from BioLegend’s Total-Seq C product line.

For the CRISPR component, customers design their sgRNAs and perform the CRISPR screening process on their cells prior to sample submission for 3’ or 5’ scRNAseq applications.

Currently, you can only perform feature barcode on either cell surface proteins or CRISPR using 3’ scRNAseq, not both. 5’ scRNAseq would allow you to perform both cell surface protein detection and CRISPR on the same cell at the same time.

scATACseq

Application highlights

• Determination of single cell open chromatin regions for understanding epigenetic and regulatory variation across the genome.
• Capture up to 10,000 nuclei per well of partitioning chip. 

Workflow - The single cell ATAC assay is used to assess the accessibility of chromatin on a single cell level. In the first step of this workflow, single nuclei are isolated from the single cell suspensions. Next, the nuclei generated in the first step undergo a transposase enzymatic reaction where accessible DNA regions are fragmented and tagged with sequencing adaptors. The transposed nuclei are then partitioned into GEMs where each nuclei is individually barcoded and made ready for library preparation.

Multiome (scATACseq and 3’ scRNAseq)

Application highlights

• Determination of single cell open chromatin regions for understanding epigenetic and regulatory variation across the genome.
• Gene expression profiling.
• Has the added benefit of scATACseq and scRNAseq obtained from the same cell.
• Capture up to 10,000 nuclei per well of partitioning chip. 

Workflow – This application has a similar workflow as the scATAC seq application. In the first step of this workflow, single nuclei are isolated from the single cell suspensions. Next, the nuclei generated in the first step undergo a transposase enzymatic reaction where accessible DNA regions are fragmented and tagged with sequencing adaptors. The transposed nuclei are then partitioned into GEMs where each nuclear material in each nuclei is barcoded. The resulting barcoded nucleic acid content is then amplified. The resultant amplified product is used for both the scATAC library generation as well as the cDNA amplification and library preparation for the 3’ scRNAseq component.

Sample requirements

Sample Type - Samples should be submitted as single cell suspensions in 1.5 mL microfuge tubes. Cell dissociation from tissue and removal of significant cell debris via filtration (e.g. 40 um Flowmi tip cell strainer) should be performed (by the investigator) prior to submission.

Formalin fixed cells are not suitable to use in either the 3’ or 5’ scRNAseq applications.

Cell enrichment - Flow cytometry, magnetic bead positive or negative cell selection, etc.. should be performed by the submitting investigator prior to sample submission.

Viability - Optimal viability is >90% however viabilities of 70-90% are acceptable. Viable cell suspensions can be cryopreserved (viability >90% prior to cryopreservation) and then submitted at a later time. Please note that the viability of previously cryopreserved samples can vary greatly. A Low viability may negatively impact the efficiency of cell capture as well as the sequencing data.

Buffer - Single cell suspensions for 3’ or 5’ scRNAseq services can be submitted in either 1X PBS (with ≤ 0.04% BSA-recommended by 10X Genomics-ideal buffer) or, for more sensitive cells, culture media. Please note that additives in media can have an effect on capture efficiency. At the minimum, EDTA/EGTA should be left out of the media since it will inhibit downstream enzymatic steps. If bringing samples in PBS, cells should have been freshly harvested (minimal transit time before submission); otherwise, cells in media may be better depending duration of the cells outside of the system or on the sensitivity of your cell suspension in a non-media environment. Fetal bovine serum is permitted in either PBS or media but should not contain more than 5% fetal bovine serum (2% or less is preferred).

scATACseq cell suspensions should be submitted in 1X PBS. We do not recommend media for either application.

Free Nucleic Acids- If samples are suspected of containing contaminating free nucleic acids (from dead, dying, or lysed cells), we recommend that you remove them from the cell suspension by low-speed centrifugation (300-500XG for 5 minutes at 4 degrees C) of the cells along with 1-2 washes with fresh PBS/media. The presence of free nucleic acids negatively impacts the efficiency of cell capture as well as the sequencing data..

Cell concentration

3’ or 5’ scRNAseq -The optimal cell suspension concentration is 1000-2000 cells/uL (100,000 total cells in 50-100 uL buffer). We can, however, run lower cell quantities (we have successfully run cells < 10,000). For low cell quantities the volume of cells required is a maximum of 10-30 uL.

scATACseq,- The cell input for this application is a minimum of 200,000 cells in 50-100 uL of PBS. Lower cell inputs are permitted in the range of 2,000-40,000 total cells in a volume of 50 uL of PBS.

Multiome (scATAC and scRNAseq)- The cell input for this application is a minimum of 200,000 cells in 50-100 uL of PBS. 

Sequencing Requirements

10X Genomics has minimum reads/cell recommendations for each application. These are very broad guidelines and may not meet the needs of your specific project. We strongly recommend that you consult your biostatistical collaborator to determine the number of cells and reads per cell required to meet your specific experimental objectives. 

The table below outlines the ATGC’s general recommendations for the number of read pairs/cell for each application. The table is meant only as a guide for sequencing coverage.

Single cell sequencing is typically performed on the Illumina NovaSeq6000 or NextSeq500 sequencers. Both sequencers allow flexibility by providing various flow cell format options to meet your sequencing requirements. The choice of sequencer and flow cell format for single cell experiments depends on the number of samples submitted and the target cell captures requested for each sample. For high sample volume or complex sample library pools, we will prescreen for appropriate sample sequencing distribution using the iSeq100 prior to sequencing on higher density flow cells.

Data Output

The ATGC only provides raw data generated from our service. Base Call files (bcl) are converted to fastq files using specific software from 10X Genomics. Modified fastq files are used for QC metrics (html files) and demultiplexing of the sequencing data using 10X Genomics Application Pipeline software. Analysis files are created using 10X Genomics Pipeline software which can be viewed using freeware analysis software which is available for download from 10X Genomics.

Please see single cell service and sequencing pricing on the ATGC Price List. ATGC Price List.

Targeted Single Cell Multi-Omics

The Mission Bio Tapestri Platform analyzes genotype and phenotype from the same single cells.

Application highlights

• Simultaneous detection of SNVs, CNVs, and protein at the single-cell level
• SNV/indel detection across 100’s of loci
• Gene and chromosome level CNV detection
• Sensitivity for rare clones- down to 0.1%
• Capture 5k-10K cells for DNA only, 2k-10K for protein
• Minimum panel: 20 amplicons, maximum 1K amplicons
• Off the shelf or custom panels
  
• Human and mouse panels

The panels are configured to run SNV alone, SNV in combination with CNV, or a triple combination of SNV, CNV, and protein detection. 

Mission Bio provides target panels for hematologic tumors, solid tumors, or the capability of custom panel creation. The ability of providing custom panels also allows for the ability to determine the status of genome editing (e.g. CRISPR/TALEN) from engineered DNA sequence edits. Specific information for each panel can be located on Mission Bio’s website link: https://missionbio.com/panels 

Technology Overview: The Tapestri microfluidics system is used to partition single cells (up to 10,000) and a protease enzyme mix into oil droplets where the cells are lysed. The resultant cell lysate partitions are then partitioned a second time into droplets that include barcoded beads, primers, and reagents for generating uniquely barcoded amplicons. Following amplification, the barcoded-DNA amplicons are used to generate Illumina compatible libraries.

During library preparation Illumina P5 and P7 sequencing adapters are added to the amplicons to facilitate sequencing on the Illumina platform. Following sequencing, the fastq files generated are analyzed using Mission Bio’s analysis software.

Sample Requirements

Sample Type: Samples should be submitted as single cell suspensions in 1.5 mL microfuge tubes. Cell dissociation from tissue and removal of significant cell debris via filtration or cell washes should be performed by the investigator prior to submission. If samples are suspected of containing contaminating free nucleic acids (from dead, dying, or lysed cells), we recommend that you remove them from the cell suspension by low-speed centrifugation (300-500XG for 5 minutes at 4 degrees C) of the cells along with 1-2 washes with fresh buffer. The presence of free nucleic acids could negatively impact the efficiency of cell capture as well as the sequencing data results. 

Formalin-Fixed cells are not suitable for use in this application. 

This technology is not currently designed for RNA transcriptome analysis. Please see 10X Genomics services for scRNAseq services. 

Cell enrichment: Flow cytometry, magnetic bead positive or negative cell selection, etc.. should be performed by the submitting investigator prior to sample submission.

Viability: Optimal viability is >90%; however, viabilities >70% are acceptable. Viable cell suspensions can be cryopreserved (viability >90% prior to cryopreservation) and then submitted at a later time. Please note that the viability of previously cryopreserved samples can vary greatly. A Low viability may negatively impact the efficiency of cell capture as well as the sequencing data. Cell nuclei protocols can be utilized for cells that have previously been frozen. 

Cell suspension buffer: 1x DPBS w/o Ca2+/Mg2+. Note: This protocol has not been validated for cells submitted in media.

Cell concentration:

Optimal: 400K total cells in a volume of 100 uL of 1x DPBS w/o Ca2+/Mg2+. (4000 cells/uL) Minimum: 200K total cells in a volume of 50 uL. 

Minimum: 200K total cells in a volume of 50 uL. 

When possible we recommended providing a higher concentration to allow for possible differences between our facility and another laboratory’s concentration measurements.

Sample QC 

Cell concentration and viability are determined using trypan blue exclusion detection and read on an automated counter. Any samples not meeting our viability requirements will also be evaluated manually under an inverted microscope where an estimate will be provided. The investigator will be able to decide at that time if they wish to proceed with processing. 

Sequencing Requirements

Sequencing requirements vary based on the panel selected.

AML panel: ~75M read pairs

Myeloid panel: ~188M read pairs

CLL panel: ~165M read pairs

THP panel: 146M read pairs

Supported sequencers that are available are MiSeq, NextSeq500, and NovaSeq6000. Most panels require 150nt paired end sequencing with 8nt dual indexes. 

Data Output: Raw data fastq files will be used for data analysis. Our bioinformatics will perform the initial analysis, which includes the read alignment, variant calling, QC metrics and loom file generation. The Loom files generated can be utilized in Mission Bio’s Tapestri Insights analysis software which will enable further data analysis, variant filtering, and data visualization. Tapestri Insights software can be downloaded from Mission Bio’s website: www.missionbio.com

Getting Started

Project Consultation: We strongly recommend a consultation meeting before starting your first Tapestri project. Consultations are free for investigators utilizing the ATGC's single cell service. To request a meeting please contact David Pollock (dpollock@mdanderson.org) or Erika Thompson (ejthomps@mdanderson.org).

Scheduling Your Experiment: The single cell service is by appointment only. You must have an appointment prior to submitting samples. Submission appointments should be made a minimum of one week in advance of your anticipated submission date. This prevents scheduling conflicts and ensures the appropriate reagents are available and at the correct temperatures for immediate use (having samples sit while we bring reagents to temperature may negatively impact data). We will make effort to accommodate appointments made 24-48 hours before submission but we cannot guarantee availability. 

To schedule a submission appointment, please contact David Pollock (dpollock@mdanderson.org). 

Sample Submission: To submit samples please complete a Mission Bio Tapestri Single Cell DNA Service Request Form and email the completed form (with an active account number and the appropriate signature) to David Pollock (see email above). In addition external submissions require a completed external billing form and an e-copy of the purchase order.