Glyoxal fixation facilitates transcriptome analysis after antigen staining and cell sorting by flow cytometry
Fig 3
RNA-seq of stained and sorted cell populations.
A: Bioanalyzer profiles of total RNA derived from unprocessed cells (harvested directly from trypsinised cells) compared to cells that have undergone glyoxal fixation, permeabilisation with 100% methanol and indirect immunofluorescence for CCNB1. 0.1–5 ng of total RNA was separated on total RNA pico chips on Agilent 2100 Bioanalyzer. B: Scatter plots comparing the normalised read count for each annotated gene in GRCh38 in poly(A)+ RNA-seq libraries derived from unprocessed and from glyoxal fixed, permeabilised and stained cells. Two biological replicates were sequenced for each condition and averaged, read counts were normalised to the total number of reads in each library. Data is shown for both COLO205 and MCF-7 cells. C: Flow cytometry density plots for MCF-7 cells labelled with anti-CCNB1 primary antibody and donkey Alexa Fluor-488 conjugated secondary antibody and sorted using a BD FACSAria III sorter. Intact cells (shown within the elliptical gate) were distinguished from off-scale events and cell debris using forward scatter (FSC) and side scatter (SSC) measurements (panel 1). Doublets were excluded from the gated cells by a 2-step gating strategy with pulse height (H) plotted against area (A) for FSC parameter followed by SSC-H versus SSC-A plot (panel 3). Fluorescence thresholds for isolation of CCNB1 positive and negative cell fractions are shown in panel 4. Gates were set using the negative control and the CCNB1 positive and negative sorting gates were moved apart from each other to maximise purity when sorted. D: Fluorescence histogram of DAPI intensities for sorted CCNB1 negative and positive populations. E: Scatter plots comparing the normalised read count for each annotated gene in GRCh38 in poly(A)+ RNA-seq libraries derived from two biological replicates each of CCNB1 positive (left) and negative (right) MCF-7 cells. Note the particularly tight correlation after sorting towards the antigen of interest. E: Hierarchical clustering analysis of the 161 significantly different expressed genes identified DEseq2 analysis (p<0.05) comparing CCNB1-positive and CCNB1-negative MCF-7 samples.