NextSeq 500 sequencing; RNA-seq of XTE cells and vesicles, XEN and XEN-like cells, and ES cells
Cells were collected from culture using standard procedures.  Mouse embryonic stem cells were maintained in the presence of 2i/Lif (0.4 µM PD0325901, 3 µM CHIR99021 and 4 ng/ml Lif), on plastic dishes coated with 0.2% gelatine.  XTE vesicles were obtained by culturing the XTE cells under non-adherent conditions for a period of 3-4 days in the XTE medium. XTE cells were obtained by either from ES cells or XEN cells or directly from the embryo by culturing them on feeders in an N2B27 basal medium containing Bmp4, CHIR99021, hFGF4, Heparin and Activin (XTE medium). The XTE cells were further selected by FACS sorting for cells expressing both PDGFRa and CD40 surface markers.  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.