Can dissociated FFPE samples be flow sorted for use with the Fixed RNA Profiling assay?

Question: Can dissociated FFPE samples be flow sorted for use with the Fixed RNA Profiling assay?

Answer: Dissociated FFPE samples can be enriched using flow cytometry and cell sorting prior to Chromium Single Cell Gene Expression Flex assay. Flow cytometry of samples provides advanced sample clean-up to remove cell debris and unwanted particles that could be carried over during FFPE curl dissociation. Additionally, DNA and RNA specific dye stains permit selective enrichment of samples that contain nucleic acid. The removal of unwanted debris accompanied with enrichment of samples containing nucleic acid may improve analysis metrics such as cell counting, usable data, complexity and clustering.

Sorting using DNA and RNA dyes

DNA and RNA dyes can be used to stain, detect and flow-sort samples that contain nucleic acids from samples devoid of nucleic acids and/or cellular debris. In our limited testing we have used Invitrogen™ DAPI and Hoechst Nucleic Acid Stains (Catalog # D1306) and SYTO® RNAselect™ Green Fluorescent Cell Stain (Catalog # S32703) to stain dissociated FFPE samples. To successfully sort samples please read: What are the best practices for flow sorting cells for 10x Genomics assays? 

FFPE sample sorting is not recommended if the user is unable to obtain at least 100,000 nuclei post-sorting. It is recommended that users optimize DNA Dye and RNA dye concentration suitable for their samples prior to conducting the actual experiment. For more accurate flow cytometry gate selection, a small aliquot of unstained sample should be set aside as a negative control. 

Staining Procedure

Nuclei staining was performed as follows:

1. Nucleic acid dyes were prepared as recommended by the manufacturer. Nuclei stain was performed using DAPI at 1 ug/mL and RNAselect™ at 5 uM final concentrations.
2.  Samples were stained with RNAselect™ for 20 minutes at room temperature in the dark. 
3. Samples were washed twice with 1 mL of ice-cold PBS + 1% BSA (nuclease free).
4. Samples were resuspended in 500 uL of PBS + 1% BSA (nuclease free) and stained with DAPI for 10 minutes on ice in the dark. 
5. Samples were washed twice with 1 mL of ice-cold PBS + 1% BSA (nuclease free).
6. Samples were resuspended in 300 uL of PBS + 1% BSA (nuclease free) and filtered using a 30-um filter prior to sorting procedure (resuspension volume should be adjusted based on nuclei concentration to provide optimal events/second and flow rate for a successful sort settings).

Flow Cytometry Gating Strategy

The gating strategy for dissociated FFPE samples should be as follows: 

1. Use an unstained sample to set the forward scatter, side scatter and singlet gates. 
2. The unstained sample should also be used to set the threshold for DAPI and RNAselect™ gates.
3. Use a stained sample to first select the DAPI positive population. 
4. Under the DAPI sub-gate select and sort RNAselect™ positive population.

Using careful gating the DAPI stained sample could be segregated to DAPILow and DAPIHigh populations. The two DAPI populations could further be examined for RNAselect™ signals.

We have observed that sorting DAPILow , RNAselect™positive population results in extremely low sample recovery. Users should collect the DAPIHigh + RNAselect™ positive population for the downstream Flex assay. 

In the above example, roughly half of the sample is DAPILow. The DAPILow population significantly lacks detectable nuclei as shown in the above table. Flow sorting removes unwanted debris and nuclei with low nucleic acid content.

Below is a comparison of data quality between unsorted (bulk) and sorted lung FFPE samples.  Addition of a flow sorting step improves several Chromium Single Cell Gene Expression Flex assay metrics and enhances data quality.

Sorting FFPE samples improves microfluidic nuclei recovery (A.) and of Flex metrics (B.). 

Flow sorting improves barcode rank plot (C.) and clustering (D.) when compared to unsorted (bulk) samples.

Products: Fixed RNA Profiling, Single Cell Gene Expression Flex