Transcriptome profiling of purified red blood cells from Nxf1 mutant and control mice [Erythrocytes]

Purpose: Nxf1 is thought to be an essential nuclear exporter of messenger RNA (mRNA) in eukaryotic cells. Whether perturbations in the Nxf1 pathway affect mammalian physiology is not known. The aim of this study is to determine the impact of a Nxf1 mutation in the representation of mRNA transcripts in erythrocytes. Methods: Red blood cells were obtained from cardiac puncture, mixed with 100µl Aster Jandl citrate-based anticoagulant and subsequently diluted in 2 ml of phosphate buffer saline (PBS) with 2mM EDTA. This suspension was layered with 1.5 ml Ficoll-paque PLUS (GE Healthcare) and centrifuged at 400g for 30 minutes at room temperature with minimal acceleration and brake. The red blood cell containing pellet was washed twice with PBS 2mM EDTA and lysed for RNA extraction. The purity of each sample was checked by flow cytometry to assess the relative contamination of Ter199+ red blood cells with CD41+ platelets, CD45+ lymphocytes and DRAQ5+ (Thermo Fisher Scientific) nucleated cells. Red blood cell suspensions used for RNAseq were > 99.8% pure. RNA was extracted with Qiagen RNA extraction kit. Erythrocytes were purified from individual Nxf1 mutant males (n=3) and three individual littermate wild-type controls (n=3).