title: NextSeq 500 sequencing; RNA-seq of XTE cells and vesicles, XEN and XEN-like cells, and ES cells
design: RNA-seq of XTE cells and vesicles, XEN and XEN-like cells, and ES cells
library_construction_protocol: Cells were collected from culture using standard procedures.  XEN-like cells were generated from ESC that carry doxycycline (Dox) inducible Gata4 transgene and Histone H2B:Venus knock in cassette in the Gata6 locus {Schroter et al., Development, 2015}. Following a  pulse of Dox driving transient Gata4 expression, the ESC were converted into XEN-like cells which Gata6-H2B:Venus reporter and acquire PDGFRα expression. Total RNA was isolated using Macherey-Nagel RNA isolation kit. 1ug of this was used for library preparation and sequencing.  Enrichment of mRNA was performed using NEBNext® Poly(A) mRNA Magnetic Isolation Module and cDNA library was constructed using NEBNext® Ultra™ II Directional RNA Library Prep Kit.