Straightforward extraction of high-quality RNA from glyoxal-fixed cells facilitates transcriptome analysis after cell staining and sorting by flow cytometry

We have developed a methodology for isolation of high-quality RNA from cells that are fixed, stained and sorted by flow cytometry that allows routine transcriptomic analysis of highly purified cell populations and single cells. This method, essentially developed by modifying existing staining and sorting protocols, involves fixation of cells with glyoxal, an alternative fixative to commonly used formaldehyde, followed by methanol permeabilization, a 2-step primary and secondary antibody staining and sorting by flow cytometry. The advantage of using glyoxal is that it does not crosslink RNA to proteins nor form stable RNA adducts, ensuring that RNA remains accessible and amenable to enzymatic manipulation after glyoxal fixation. The dataset comprising mRNA seq libraries from unprocessed or fixed and stained human cancer cells demonstrate that RNA recovered from glyoxal-fixed cells does not retain sufficient glyoxal adducts to impair reverse transcription, and also reveal very few differentially expressed genes between the 2 groups. The dataset derived from fixed and stained cells that were sorted into CCNB1 positive or negative fractions show the applicability of this method for sorting highly purified cell fractions as the CCNB1 positive fractions show strong enrichment for G2-phase cells according to the GO analysis. RNA-seq libraries from COLO205 and MCF7 human cancer cells that were unprocessed or fixed and stained for CCNB1, and from MCF7 cells that were stained and flow sorted into CCNB1 positive and CCNB1 negative fractions.