Quantification of transcriptional changes during in vivo mouse erythropoiesis by nascent RNA-sequencing

We isolated erythroid progenitor cells at various stages of differentiation from mouse fetal liver using flow cytometry. We metabolically labelled RNA from purified progenitor populations with 4-thiouridine (4sU) for 45 minutes in ex vivo culture, then extracted cellular RNA. Nascent RNA was biotinylated by reaction of the incorporated thiol groups with MTS-Biotin, then purified using streptavidin nanobeads and sequenced. Overall design: Nascent RNA-sequencing of metabolically (4sU) labelled RNA in primary erythroid progenitors from mouse fetal liver