title: NextSeq 500 sequencing; RNA-Seq to investigate the role of membrane-to-cortex-attachment in mESc pluripotency
design: RNA-Seq to investigate the role of membrane-to-cortex-attachment in mESc pluripotency
library_construction_protocol: To induce formation of embryoid bodies (EBs) mECs originating from Serum+LIF monolayer culture were seeded on non-treated (i.e. non-adhesive) culture-dishes at a density of about 80.000/square cm in medium without LIF to allow for spontaneous differentiation. Expression of constructs was induced by adding 1ug/ml doxycycline at the time of seeding. EBs were grown for 96h. Media was exchanged after 48h.  1e5 to 1e7 cells were pelleted via centrifugation and total RNA was extracted with the mirVana miRNA Isolation Kit (Thermofisher) according to the manufacturers recommendations. Barcoded stranded mRNA-seq libraries were prepared from high quality total RNA samples (about 250 ng/sample) using the NEBNext Poly(A) mRNA Magnetic Isolation Module and NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England Biolabs (NEB), Ipswich, MA, USA) implemented on the liquid handling robot Beckman i7. Obtained libraries that passed the QC step were pooled in equimolar amounts; 2.2 pM solution of this pool was loaded on the Illumina sequencer NextSeq 500 and sequenced uni-directionally, generating ~500 million reads, with an average read length of about 80 bases.