Applying cellular fixation and cell-type isolation for single-cell sequencing

Amplexa has supported Prof. Ditte C. Andersens-group in applying and validating a novel technique for cell isolation for single-cell sequencing that allows the samples to be stored at -80 degrees for a prolonged period before further single-cell library preparation. Fixation, combined with viability staining and Fluorescence-activated cell sorting, facilitates researchers to apply high-resolution single-cell RNA sequencing (scRNA-seq) technologies on specific cell types of interest for multiple time points. The technique allows transportation of samples to other facilities should methods for library preparation (e.g., 10x Genomics Chromium) not be available in-house..

Most experiments that apply single-cell RNA sequencing rely on fresh tissue processed for library preparation immediately after collection, which introduces logistical concerns and limitations in study design. While the standard preparation technique enables the analysis of multiple cell populations, it can limit the transcriptomic coverage of rare cell types. We present the application of Methanol for cellular fixation in combination with FACS to enrich rare cell populations based on specific surface markers for scRNA-seq library preparation. The protocol had a strict focus on preserving RNA integrity.

The protocol was validated by comparison with fresh material and tested in multiple species, including zebrafish, by applying scRNA-seq pipelines developed at Amplexa. The protocol produced scRNA-seq data for highly enriched, heterogeneous cardiomyocytes. Further, ploidy stratification allowed the identification of differentially expressed genes in tetraploid cardiomyocytes before and after birth in mice, as well as enriched transcription factors.