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 cells were derived from blastocyst by plating E3.5 embryos into individual wells of 96-well plate with feeders and cultured in DMEM medium containing 15% FBS, 2 mM L-glutamine, 1 mM sodium pyruvate, 0.1 mM non-essential amino acids, 50 U/ml penicillin-streptomycin, 0.1 mM 2mercaptoethanol. After 4 days the blastocyst outgrowths were trypsinised and transferred to new wells to allow the emergence of XEN cells. 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.