Illumina HiSeq 2500 sequencing; NEUROMESO
NEUROMESO
Mouse ES cells were grown as monolayer in N2B27 medium supplemented with 10ng/ml bFGF (R&D) for 3 days (D1-D3) and then were transferred into serum free media without bFGF (D3-D5).In these conditions cells acquired and anterior neural identity (NA). To induce ventral hindbrain identity NPCs (NH), 100nM RA (Sigma) and 500nM SAG (Calbiochem) was added from D3-D5. Spinal cord identity (NP) was induced by the addition of 5nM CHIR 99021 (Axon) from D2 to D3 followed by 100nM RA, 500nM SAG from D3-D5. To induce mesodermal differentiation the cells were treated with 5uM CHIR99021 from D2-D5 (Meso). RNA samples were collected each day of differentiation from D1 to D5 for the different conditions (NA,NH,NP and Meso). Total RNA was processed according to the TruSeq protocol (Illumina). Three separate read RNA libraries (biological replicates) were barcoded and prepared for each time point. Library size, purity and concentration were determined using Agilent Technologies 2100 Bioanalyzer.
Experimental Factor: time
2 day