NextSeq 500 sequencing; RNA-Seq to investigate the role of membrane-to-cortex-attachment in mESc pluripotency
RNA-Seq to investigate the role of membrane-to-cortex-attachment in mESc pluripotency
To induce differentiation and the exit from pluripotency mECs originating from 2iLIF culture were plated on gelatine-coated culture-dishes at a density of about 40.000/square cm in plain N2B27 medium (or 2iLIF medium as control) and cultured for 48h. Expression of constructs was induced by adding 1ug/ml doxycycline at the time of seeding.  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 TruSeq Stranded mRNA Library Prep Kit (Illumina, San Diego, CA, USA) implemented on the liquid handling Beckman Fx robot. 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."