10X Chromium FLEX (fixed RNA)

Single-cell profiling of gene expression levels on fixed cells

In the past, most single cell assays required live cells and thus, could make the logistics challenging. However, with the introduction of the 10X Genomics FLEX assay, users are now able to fix and store their samples before further processing, leading to an increased flexibility in the lab and experimental planning. 

IMPORTANT! FLEX is only compatible with human and mouse tissue. 

FLEX projects can be ordered via iLab (if the project is to be run at NGI Stockholm, search for the ESCG facility) or via the NGI order portal (if the project is to be run at NGI in Uppsala).

Since the FLEX assay is a probe-based method (transcriptome-wide), only endogenous genes (which are included in the probe set; non-coding genes are not covered) can be detected. In order to detect any exogenous genes, custom probes have to be designed and included (not supported by 10X Genomics, but guidelines can be found here). A full list of probes can be found here. Information about SNVs or isoforms is lost during the assay since only ligated probes are sequenced. For the same reason, FLEX can not yield information such as V(D)J or guide RNA sequences. 

Options to multiplex your samples are available, either to analyse multiple samples in parallel or to increase the cell number for a single sample. Multiplexing will decrease the per sample cost or cost per cell, especially if running many samples/cells. Discuss with us if you are interested in that option. 

Sample requirements

  • Start with high quality samples (>80% viability, free of debris). The 10X FLEX assay is robust even for samples with lower viability (50%) but lower viability samples may have more variable cell calling and lower sensitivity. Sample quality is directly correlated to data quality.
  • The maximum recommended cell size is 30 µm, larger cells may clog the microfluidic channels of the 10X Genomics chips. If cells are > 30 µm, nuclei isolation should be performed.
  • We recommend starting with 1 x 10^6 cells for fixation whenever possible. For sample fixation, the recommended minimum of fresh cells or nuclei suspensions is 300,000 cells or 500,000 nuclei, respectively.
  • For proceeding with probe hybridization we require a minimum of 200,000 cells or 400,000 nuclei for single-plexed samples. We recommend delivering more than this to ensure there is enough for washing and counting. 
  • Samples need to be fixed following the 10X Genomics protocol for long-term storage. 

Compatible sample types

Multiplexing

FLEX reagents can be purchased in three different variants:

  • Single-plex assay: Each probe has one sample-specific barcode. This kit can be used to run one sample per FLEX reaction.
  • 4-plex kit: The kit contains 4 sets of probes with each having a sample-specific barcode. Up to 4 samples can be multiplexed per reaction.
  • 16-plex kit: The kit is the same as the 4-plex kit but with 16 distinct sample-specific barcoded probes instead of 4. 

* These cell numbers are not supported.

Source: CG000527_ChromiumFixedRNAProfiling_MultiplexedSamples_UserGuide_Rev_E.

Illustration of different multiplex options to either maximize the number of samples or the number of cell analyzed per FLEX reaction. Source: CG000527_ChromiumFixedRNAProfiling_MultiplexedSamples_UserGuide_Rev_E.

Feature barcode technology for protein detection

The FLEX protocol is compatible with both TotalSeq-B and TotalSeq-C antibodies (different kits are required). Proteins need to be labelled with oligonucleotide-conjugated antibodies prior to sample fixation. Antibodies for protein staining need to be purchased separately, e.g. from BioLegend.

Source: CG000527_ChromiumFixedRNAProfiling_MultiplexedSamples_UserGuide_Rev_E.

Starting up a FLEX project with NGI

  • Contact us to discuss your project. 
  • Read our user guidelines (NGI Stockholm)
  • If possible, perform a pilot of the sample preparation and fixation to ensure the required sample quality and quantity is achieved.
  • Fix your samples following the 10X Genomics protocol for long-term storage. Please note that you will need to purchase all required reagents for fixation yourself, including the Chromium Next GEM Single Cell Fixed RNA Sample Preparation Kit.
  • Store the samples after fixation at -80ºC, in clearly labelled tubes. All samples should be shipped on dry ice.

Information we need prior to sample delivery

  1. Once you have placed your project request in iLab (if the project should be run at NGI Stockholm) or via the NGI order portal (if the project should be run at NGI in Uppsala), a project coordinator will set up your project. Please make sure it reflects your specifications and if everything looks like it should please agree to the terms and conditions electronically.
  2. Return the filled-in safety declaration (if applicable) along with the user agreement. 
  3. We will prepare sample-specific barcodes for the project. Attach them to low-bind 1.5 ml tubes before freezing your samples.

What we do with your samples

Once your samples arrive at NGI, we start by counting your cells using a fluorescent dead/live stain to make sure that the samples meet our requirements. You may choose to be present during counting.

If the samples fail this quality control step, we will only continue with the samples if we have received written confirmation, prior to sample delivery, from you that you would like to continue with the samples no matter the counting outcome. In this case, we will charge you for all of the 10X reactions that were used. If it was decided to not continue with your samples, we will not charge you anything. 

If the samples pass the reception control, we will continue with the rest of the protocol.

Library preparation

The library preparation consists of several steps:

  1. Probe hybridization

Left-hand side (LHS) and right-hand side (RHS) probes are hybridized to complementary RNA molecules.

  1. GEM generation and barcoding

Unhybridized probes are washed away before creating an emulsion containing barcoded gel beads and cells. Cells are counted before the Gel Bead-in-Emulsion (GEM) generation is performed on the Chromium controller.

  1. GEM recovery and pre-amplification

The emulsion is broken by adding a recovery agent and ligated probe pairs are pre-amplified.

  1. Fixed RNA – Gene expression library construction

Each sample receives a sample-specific index during a final round of amplification.

Library QC and sequencing

In this step, we evaluate the yield and size distribution of the libraries and inform you about the QC status of each sample. Once the libraries have passed this QC step, they are queued for sequencing. The sequencing will be carried out following the setup stated in the agreement.

Bioinformatics

Basic bioinformatic analysis using the CellRanger software from 10X Genomics.

Last Updated: 25th January 2024

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