title: Illumina HiSeq 2500 sequencing; RNAseq libraries to study early developmental progenitors (24hrs differentiation vs hESCs)
design: RNAseq libraries to study early developmental progenitors (24hrs differentiation vs hESCs)
library_construction_protocol: Illumina TRUseq method to generate highly strand-specific next-generation sequencing (NGS) libraries H9 human ESCs (H9 line) were cultured in mTeSR1 medium on Matrigel-coated plates. Colonies were passaged for maintanence by Gentle Cell Dissociation Reagent  Primitive streak differentiation was induced by dissociation of colonies into single cells using accutase and subsequently seeding them on Matrige-coated 6-well plates containing mTESR1 medium  supplemented with ROCKi at 100% confluency. Next day cells were washed with PBS, and the medium was changed to RPMI1640 medium supplemented with L-Glutamine, B27 without insulin, 10µM ROCK inhibitor and 10µM CHIR99021 or alternatively 200ng/ml of purified WNT3A recombinant protein. For trophoblast directed differentiation human ESCs were passaged using 0,25% Trypsin-EDTA and plated as single cells (105,000 cells/cm2) on MG coated plates in DMEM/F12 medium, supplemented with 20% KnockOut Serum Replacement, Glutamax, nonessential amino acids, beta-mercaptoethanol, and 50 ng/ml BMP4. Medium was changed daily. RNA-seq
attributes.tag: Experimental Factor: growth condition
attributes.value: undifferentiated