10X Genomics 3’ or 5’ gene expression (GEM-X or Next-GEM)

Profiling of gene expression levels at single-cell resolution.

10X Chromium offers whole transcriptome profiling at the single cell level. In the partitions generated in the emulsion, transcripts are captured by oligos on 10X gel beads and receive both a cell-specific barcode and a UMI during reverse transcription.

Transcriptome analysis of single cells using 10X chromium can be performed using either the 3’ or the 5’ kits. The most significant difference between these kits is which end of the transcript is captured onto the gel beads. For 3´ gene expression (GE), the gel beads contain poly(dT) oligos to capture the 3’ end of the transcripts whereas for the 5’ GE beads the 5’ end of the transcripts are captured by a TSO. 

If you are interested in running your 3’ or 5’ GE experiment at NGI, please contact us at support@ngisweden.se.

RNA capture and cDNA synthesis using the 10X single cell 3’ GE kit (left) and the 10X 5’ GE kit(right). Adapted from https://cdn.10xgenomics.com/image/upload/v1710230393/support-documents/CG000731_ChromiumGEM-X_SingleCell3_ReagentKits_v4_UserGuide_RevA.pdf & https://cdn.10xgenomics.com/image/upload/v1710231087/support-documents/CG000733_ChromiumGEM-X_SingleCell5_ReagentKitsv3_UserGuide_RevA.pdf 

Compatible sample types

The Chromium 3’ and 5’ GE methods are compatible with cell or nuclei suspensions from diverse species.

Sample requirements

In order to obtain high quality data from the experiment the sample should have:

  • No or minimal amounts of cell debris.
  • No or low levels of cell/nuclei aggregates.
  • High cell viability of >90%. Samples with a slightly lower viability (>80%) are acceptable but data quality might be negatively impacted.
  • For nuclei: Intact nuclei with high-quality nuclear membranes and <5% of intact cells.
  • A concentration of 700-1,200 cells or nuclei per µl for a targeted cell recovery of 500-10,000 cells or a concentration of 1,300-1,600 cells or nuclei per µl for a targeted cell recovery of 10,000-20,000 cells.
  • A maximum cell size of 30 µm. Larger cell sizes might lead to clogging of the microfluidic channels. If you have cells larger than 30 µm, it is recommended to isolate nuclei.

We always recommend performing a trial prep for any new sample types before handing over any samples to NGI. The sample quality must be assessed before sample delivery. For nuclei, we recommend to assess nuclei quality at 60x in a microscope.  

Extra options

Chromium 3’ and 5’ gene expression is compatible with Feature Barcoding/Cell surface protein detection.

5’ Gene expression also offers the option of Immune profiling (V(D)J analysis) for human or mouse samples, BEAM antigen repertoire mapping, as well as CRISPR analysis.

V(D)J Immune Profiling – for 5’ (GEM-X)

V(D)J

The Single Cell 5′ protocol offers solutions for measuring immune repertoire information and gene expression from the same cell. Profile full length (5′ UTR to constant region), paired T-cell receptor (TCR), or B-cell receptor (BCR) transcripts from the same cell. 

How it works

After generating the full-length cDNA using the 10X Chromium 5’ GE kit, the 10X barcoded, full-length V(D)J segments are enriched using primers specific to the TCR or BCR constant regions. The resulting, enriched sample is used to prepare dual-indexed sequencing libraries, in which enzymatic fragmentation creates variable size fragments that collectively cover the V(D)J region. 

Sample and method compatibility

Immune profiling is compatible with both cell and nuclei samples from mouse and human. If both T-cells and B-cells are expected to be present in the sample, it is possible to create libraries for both, resulting in one GE library, one BCR library and one TCR library from the same sample. 

In addition, Immune profiling can be combined with feature barcode technology for cell surface protein and immune receptor mapping as well as CRISPR screening.

CRISPR analysis – for 3′ (next-GEM) or 5′ (GEM-X)

CRISPR

Single cell CRISPR screens can be used to study genetic regulatory networks. Using 10X Genomics’ technology CRISPR-mediated perturbations can be read out alongside a cell’s gene expression at single cell resolution. 

We offer both Single Cell 5′ CRISPR Screening (GEM-X and Next-GEM) and Single Cell 3′ CRISPR Screening (Next-GEM). The choice of method will primarily depend on whether you would like to use GEM-X (recommended) or Next-GEM, and whether the assay should be combined with VDJ sequencing and/or cell surface protein detection. 

From: CG000398_ChromiumSingleCellCRISPR_Screening_Expt.PlanningGuide_Rev_C.pdf

IMPORTANT! It is crucial to test the guide efficiency and to determine the guide frequency of each sgRNA in the pool before starting any single cell experiment.

Sample requirements

Same as for 3’ and 5’ GE.

10X Genomics recommends screening 100-200 cells per targeting guide RNA and 500-1000 cells for non-targeting guide RNAs.

Guide compatibility with single cell 5′ CRISPR assay

From: CG000398_ChromiumSingleCellCRISPR_Screening_Expt.PlanningGuide_Rev_C.pdf

Recommended reading: 10X Genomics CRISPR Experiment Planning Guide

Feature barcoding (eg. for hashing) – for 3′, 5′, flex

Feature barcode detection

Both the 3’ GE and 5’ GE kits offer the possibility of capturing feature DNA barcodes introduced before the GEM generation. Feature barcoding can be used for multiple purposes (see table).

Feature barcoding techniques and compatibility

Feature barcode usagekit compatibility (GEM-X)
Antibody capture for cell surface protein3’, 5’, FLEX*
Sample multiplexing (“hashing”)3’ and 5’
CRISPR guide capture5’
Antigen Capture (BEAM)5’
* FLEX is based on Next-GEM reagents

How it works

After GEM generation the feature barcode is captured on the 10X gel beads and receives the same 10x barcode as the cDNA from the cell. 

After pre-amplification, the sample is used both for creating the GEX library and an enriched feature barcode library. Both libraries are sequenced and the information reconciled by the common cell specific barcode, allowing for tracking presence of the feature oligo back to the specific cell and its expression pattern.

The manner of capturing the feature barcode oligo on the beads differs between the 3’ kit and the 5’ kit.  

The 3’ gel beads contain, in addition to the poly(d)T for capturing RNA, capture sequence 1 and are compatible with feature barcode oligos containing the complementary sequence.

For the 5’ kit, the feature barcode oligo must contain a capture sequence complementary to the TSO on the gel bead. 

Example of feature barcode in 3’ GE. The feature barcode oligo is attached to an antibody specific to a cell surface protein. Once in the single cell emulsion the feature barcode gets captured on the bead by capture sequence 1.  Transcript extension during the same step as cDNA synthesis leads to incorporation of the cell specific 10X barcode. In the 5’ kit the feature oligo gets captured by the TSO. Adapted from: https://cdn.10xgenomics.com/image/upload/v1710230668/support-documents/CG000732_ChromiumGEM-X_SingleCell3_ReagentKitsv4_CellSurfaceProtein_UserGuide_RevA.pdf

Cell surface protein

If the feature barcode oligo is coupled to an antibody specific for a cell surface protein of interest, including it in the 10x GE experiment will allow for distinguishing cells in the sample that express this particular protein and connect it with the GE profile.

Sample multiplexing using antibodies

Labeling cells with antibody-oligonucleotide conjugates can also be used as a means for sample multiplexing (also called “hashing”). For such experiments, the different cell samples are labeled with antibodies specific for a common cell surface antigen conjugated to oligos with different barcodes before the 10x experiment. The sequences from the feature barcode library (or HTO library) are used for demultiplexing the samples after sequencing.

In addition to allowing for combining multiple samples in the same 10x reaction, the same technique can be used to increase the number of cells in a single reaction by aiding in identification of multiplets. If a single cell suspension is split into 2 or more tubes and labeled with different antibody-oligonucleotide conjugates, droplets that receive more than 1 cell can be distinguished by the presence of more than 1 feature barcode. Please note that this only allows the identification and filtering of the multiplets, it cannot be used to resolve the data from the individual cells in the multiple droplet. Please note that using feature barcoding for hashing purposes is not officially supported by 10X. 

Recommended antibodies

10x fully supports cell surface protein detection using the Totalseq B or Totalseq-C antibody oligonucleotide conjugates from Biolegend. Use of Totalseq-A is no longer officially supported by 10x and therefore no longer offered by NGI. 

Totalseq-B is compatible with the 3’ GE kit and Totalseq-C is compatible with the 5’ GE kit. 

NGI normally has a number of Totalseq-B and -C conjugates for sample multiplexing of human and mouse cells, and offers users to buy aliquots. For ordering other compatible conjugates or designing custome conjugates, we recommend contacting Biolegend.

It is also possible to conjugate oligos to other antibodies of interest. For more information please have a look at the this 10x support page

Please note that all labeling is performed before delivering the sample to NGI. Please refer to the 10x protocol Cell Surface Protein Labeling for Single Cell RNA Sequencing Protocols with Feature Barcode technology

We recommend testing the protocol to generate your antibody-labelled single cell suspension before scheduling your run. 

Antigen Capture – BEAM

The Chromium Single Cell 5′ Barcode Enabled Antigen Mapping (BEAM) protocol makes it possible to map antigen-specificity of B or T cells while simultaneously obtaining cell surface gene expression profiles and immune receptor sequences for the same cell. 

In BEAM the antigens of interest are labeled and conjugated to feature barcode oligos (the “BEAM conjugate”) and introduced to the cell suspension. The population of immune receptors specific to the antigen bind the conjugate. The cells are also stained with Immunecell panels of labeled antibodies, allowing for FACS sorting of antigen specific B or T-cells. 

The cells are then processed using the 10X Chromium 5’ kit, generating a GE library, an antigen capture library and a V(D)J library for sequencing. The method is also compatible with cell surface protein detection. 

Figure outlining the principle of BEAM B-cell isolation. For T-cells the BEAM conjugate includes MHC monomers and the antigenic peptide must first be mixed with the BEAM-conjugates since the TCR only recognizes peptides in the context of the MHC. Image from https://www.10xgenomics.com/support/software/cell-ranger/latest/getting-started/cr-5p-what-is-antigen-capture

Starting a 3’ or 5’ GE project with NGI

  • Contact us at support@ngisweden.se to discuss your project.
  • Read our current user guidelines here (v5).
  • Submit a project request through the iLAB system (search for the ESCG facility). A project coordinator will set up your project and get back to you. Please check the project setup to make sure it reflects your specifications and if everything looks correct, agree to the terms and conditions within iLAB.
  • If possible – run a pilot for the sample preparation to ensure that it yields high quality cell suspensions/nuclei.
  • Book a day/days to bring your samples to us.

Information we need prior to sample delivery

  1. An electronically accepted project request in iLAB, with no remaining questions about setup or number of samples.
  2. A filled-in safety declaration that is sent to you along with the user agreement (if applicable).

What we do with your samples

Upon delivery of the samples to NGI, the responsible lab staff will count the cells and check the quality of the cell suspension before you leave.

Should the sample be sub-optimal and you still wish to continue with the experiment, we will ask you to confirm this in writing. In such a situation you will be charged the cost of the 10x reactions regardless of the outcome. If you decide not to continue, there will be no charge unless a kit was purchased specifically for you (communicated to you before setting up your project).  

All sample manipulation such as labeling, multiplexing etc must be performed by the researcher prior to delivery of the sample. The delivered sample should be ready to load on the 10X Chromium instrument. 

Library preparation

1. GEM generation, barcoding and RT

The samples are loaded on the Chromium X controller for GEM generation and cDNA synthesis inside the GEMs. During cDNA synthesis, a cell-specific barcode is incorporated into the full-length cDNA inside the GEMs.

GEM generation. Adapted from https://cdn.10xgenomics.com/image/upload/v1710230393/support-documents/CG000731_ChromiumGEM-X_SingleCell3_ReagentKits_v4_UserGuide_RevA.pdf

2. Post GEM-RT cleanup, cDNA amplification and cDNA QC

The emulsion is broken by the addition of a recovery agent and the first-strand cDNA is purified before cDNA pre-amplification. The amplified cDNA is purified and the quality is checked by BioAnalyzer.

3’ GE cDNA amplification (top). Representative trace of amplified 3’ GE cDNA (bottom). Adapted from https://cdn.10xgenomics.com/image/upload/v1710230393/support-documents/CG000731_ChromiumGEM-X_SingleCell3_ReagentKits_v4_UserGuide_RevA.pdf

3. cDNA purification and library preparation

The cDNA is enzymatically fragmented and indexed to produce the final sequencing library.

4. Library QC and Sequencing

The library is QCed by Qubit, BioAnalyzer and qPCR before pooling for sequencing.

Representative trace of gene expression library (top). Chromium single cell 3’ gene expression library structure (middle). Chromium single cell 5’ gene expression library structure (bottom). Adapted from https://cdn.10xgenomics.com/image/upload/v1710230393/support-documents/CG000731_ChromiumGEM-X_SingleCell3_ReagentKits_v4_UserGuide_RevA.pdf Chromium single cell 5’ gene expression library. & https://cdn.10xgenomics.com/image/upload/v1710231087/support-documents/CG000733_ChromiumGEM-X_SingleCell5_ReagentKitsv3_UserGuide_RevA.pdf 

Bioinformatics

Basic bioinformatic analysis using the CellRanger software from 10X Genomics. Your data will be delivered to you through the DDS online delivery system.

Last Updated: 21st May 2024

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