An overview of the new fixed RNA (“Flex”) assay by 10X Genomics Science & Technology Advisor Dr. Egon Ranghini in the UWBC Bio-Tech Talks seminar series, 12/15/2022.
The 10X Genomics Flex Gene Expression assay, formerly called Fixed RNA Profiling, was introduced in 2022 as a way to prepare single cell/nuclei RNA-seq libraries from formaldehyde-fixed cells and tissue as well as FFPE tissue blocks. This process allows researchers to lock in the biological state of their samples at the time of fixation and store the fixed cell suspension for up to 12 months at -80C. This page is intended to help GEC customers understand the key features and considerations of this assay that distinguish it from 10X’s other fresh cell assays. Please note that for reasons discussed below, this assay is currently only officially supported for human and mouse samples. If you wish to try using this assay for similar species (e.g. rat or non-human primate), please be aware that the ability to detect those species’ transcripts may be extremely limited.
Please click here to return to our main 10X Genomics services page, or click here to visit our 10X FAQ page for additional resources and answers to some common questions about these assays.
In 10X’s traditional fresh cell assays, transcripts are captured by the 10X Genomics gel beads either at the 3′ end (by a poly(dT) capture sequence that binds any polyadenylated RNA) or by the 5′ end following a template-switching reaction. These common features allow these assays to be mostly species-agnostic. By contrast, because the fixation process is expected to lead to a certain amount of RNA degradation, the fixed RNA assay uses pairs of sequence-specific probes to bind transcripts for approximately 18,000 genes for human and about 19,000 genes for mouse (hence the species restrictions for this assay). These probe pairs bind adjacent sites on their target transcripts, are ligated together, and then become the substrate for library construction. This is the same approach used for their Visium for FFPE spatial transcriptomics assay. It is possible to design and spike in custom probes if you are interested in detecting transcripts that are not included in the standard probe sets (e.g. for viral transcripts in models of infection).
For GEC projects, the submitting lab will be responsible for the sample fixation, including the purchase of the sample fixation kit from 10X Genomics. This kit has a list price of $800 and contains reagents for the fixation of up to 48 samples (10X Genomics product #1000781). 10X Genomics provides protocols for fixing cells or tissue OR preparing a cell suspension from FFPE blocks, which are linked here. For fixing tissues or cells, we recommend the overnight fixation if possible. Some general notes for each type are:
- Cells/Nuclei: The easiest to work with, an already-prepared cell suspension can be fixed and then stored at -80C for at least twelve months. It is very important that you document the cell number and viability of your samples prior to fixation, preferably with images from a hemocytometer, automated cell counter, etc. This information is essential if any troubleshooting is needed. 10X also provides a separate protocol specifically for fixation of blood for isolation of PBMCs.
- Tissue: To maximize the amount of time this material can be stored, the recommended method is to fix the tissue and then dissociate the fixed tissue into a single cell suspension, which can then be stored long-term. The protocol also includes options for shorter-term storage of the fixed tissue if time constraints do not allow you to do the sample dissociation immediately following the fixation.
- FFPE blocks: 10X provides guidance for preparing a nuclei suspension from FFPE tissue sections either by pestle dissociation or with the use of the Miltenyi gentleMACS Octo Dissociator tissue dissociation system. The dissociated cell suspension can then be prepped for long-term storage as with the other types, if desired.
Please see this page for additional sample preparation guidance.
In the new GEM-X version of the Flex protocol, 10X Genomics states that as few as 25,000 cells can be put into the fixation reaction (with a maximum of 10,000,000 cells). Where possible, please aim for 300,000+ fixed cells. The protocol contains several wash steps that require spinning the cells down, and very low cell numbers carry a much greater risk of loss of the sample since there will likely not be a visible pellet during those steps.
Both the singleplex (for only four samples) or multiplex (up to 16, 64, or 256 samples) support feature barcoding for simultaneous protein detection. Generally speaking, this must be done before the full fixation reaction. For users interested in the intracellular protein option, please see 10X’s demonstrated protocol which includes a brief fixation step to permeabilize the cells for this prior to the full fixation reaction. In the GEM-X version of the assays, both the singleplex and multiplex options require TotalSeq C antibodies for this. Unfortunately with the change to the TotalSeq C-specific format, the kit required to actually prepare the libraries for the antibody tags is only available in a 64-reaction format (compared to the 16-reaction format available in previous versions) which carries a list price of $2,200. Please be aware of this additional cost (in addition to the costs of the antibodies and related consumables from BioLegend) when planning your experiment.
Sample multiplexing allows users to label discrete cell populations with a sample-specific barcode so that these samples can be pooled into a single GEM droplet reaction, reducing the number of reagent units needed for the experiment and typically lowering the overall cost. The most common method of doing this in the fresh cell assays involves tagging the cells with an antibody conjugated to an oligonucleotide barcode, meaning the cells must undergo additional treatment, incubation, and wash steps between the initial preparation of the single cell suspension and the partitioning and capturing of the cells in the 10X Genomics instrument.
By contrast, multiplexing in the Flex assay is built in by the inclusion of sample barcodes in the transcript-detecting probes. The reagents are sold in multiple quantities:
- The four-reaction singleplex kit. This kit includes four units of gel beads and four reactions’ worth of “barcode 1” probes. As all of the probes have the same barcode, no probe-based sample multiplexing is possible with this kit.
- 4-plex kits, which contains four units of gel beads and four different probe barcode sets (barcodes 1-4). Since only four units of gel beads are included, getting the full value of this kit requires having enough of your samples to fully multiplex all four GEM reactions.
- 16-plex kits (16 sets of probe barcodes), which are available in 64-sample format (4 units of gel beads * 16 samples pooled per gel bead unit) and 256-sample format (16 units of gel beads * 16 samples pooled per gel bead unit). Also sold as an “n-plex” kit (16 gel bead units * 16 probe barcodes) with the ability for variable pooling sizes.
When planning your experiment and sample collection, please note that with the new GEM-X version of the assay, the 16-sample multiplex Flex kit is now cheaper than even two four-sample singleplex kits.
Note that this does not necessarily require you to come up with as many as 16/64/256 unique samples. Assuming the samples have enough fixed cells to support this, cells of the same sample can be put into multiple hybridization reactions to capture more cells for those samples.
