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 Fixed RNA Profiling assay, also called the Single Cell Gene Expression Flex assay by 10X Genomics, 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 at least 6 months at -80C (per 10X Genomics internal testing as of January 2023). 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 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.
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.
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 costs approximately $400 when ordered directly from 10X Genomics, or $500 from ShopUW+, and contains reagents for fixing up to 16 samples (10X Genomics product #1000414). 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 six months.
- 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 cell 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.
Because the fixation and probe hybridization steps involve numerous washes and centrifugation steps during which we will lose cells, please be aware that the recommended number of starting cells is much higher than the fresh cell assays. Where we would normally ask for a minimum of around 100,000 cells for those assays, 10X lists a required input of at least 300,000 cells or 500,000 nuclei for fixation, with a ceiling of 10,000,000 cells/nuclei.
We can accept shipped fixed cell samples from external clients; please e-mail us at gecinfo@biotech.wisc.edu to discuss this prior to sending samples. You may ship fixed dissociated cell samples on dry ice to:
Sandra Splinter BonDurant
UW Biotechnology Center, Rm 2330
425 Henry Mall
Madison, WI 53706
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 potentially lowering the overall cost. In the fresh cell assays, this is accomplished by tagging the cells with either an antibody or a lipid conjugated to these oligonucleotide barcodes, 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 three different reaction quantities:
1. The four-reaction single-plex 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 sample multiplexing is possible with this kit. This kit is also the only version currently compatible with cell surface protein detection/CITE-seq approaches, for which the cells must be stained prior to fixation.
2. The 16-reaction multiplex kit. This kit includes four units of gel beads and four sets of probes with unique barcodes, allowing you to pool four samples per GEM reaction. Because there are only four gel bead units, you are essentially required to pool four samples per reaction in order to get the full value out of the kit.
3. The 64-reaction multiplex kit. This kit includes four units of gel beads and 16 sets of probes with unique barcodes, allowing you to pool up to 16 samples per GEM reaction. As above, the way the kits are sold means that you are required to fully multiplex this assay to get the full value of the kit.
Note that this does not necessarily require you to come up with as many as 16 or 64 unique samples. We can also put cells of the same sample into multiple hybridization reactions to capture more cells for a given sample or samples.
