RNA sequencing of Hes5.3+ cells from E5 chick embryonic retinas in control and retinoic acid treated conditions.

Two E5 (HH26) chick embryonic retinas were dissected from two embryos, electroporated with Hes5.3-GFP reporter plasmid, and incubated in 2.5 μM retinoic acid (RA) in culture medium (DMEM, 10% FBS, 1% Penicillin/Streptomycin) for 8 hours. The corresponding opposite retinas from the same embryos were used as controls and incubated in culture medium with DMSO. Cells were dissociated and GFP+ cells were sorted by FACS. RNA was extracted using TRIzol reagent, according to manufacturer's protocol. RNA samples were quantified by Qubit 4 fluorometer (Thermo Fisher), and RNA quality was assessed by BioAnalyzer (Agilent). RNA sequencing was performed. This protocol was done in triplicate for RA and control conditions. Gene expresson by RNA-seq in Hes5.3+ cells from two E5 chick embryonic retinas taken from two different embryos and treated with RA, vs two control retinas from the opposite side of the same embryos, in triplicate.