GSM4120727: NC RNAseq HH9 rep1; Gallus gallus; RNA-Seq
For isolation of neural crest cells, embryos were transfected with the enhancer reporter construct PTK-AP2aE1-eGPF. To obtain neural crest cells from different stages, embryos were cultured until HH6 (8 hours) and HH10 (15-16hours), and screened for robust GFP expression in neural crest cells. Embryo heads were dissected and incubated in Accumax (Accutase, SCR006) cell dissociation solution, for 40 minutes at RT under mild agitation. At least 5,000 GFP+ cells from each stage were sorted directly into 100uL of lysis buffer from the RNAqueous-Micro Total RNA Isolation Kit (ThermoFisher, AM1931). Total RNA was isolated according to the kit's protocol. RNA was poly(A) selected using the NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs, E7490). TruSeq-barcoded RNAseq libraries were generated with the NEBNext Ultra II Directional RNA Library Prep Kit (New England Biolabs, E7760) and sequenced on an Illumina NextSeq500 instrument. Neural plate border/neural crest dissections were performed on HH6/HH9 embryos (10 embryos/ 20 neural folds per experiment). Cells were dissociated in Accumax (Accutase, SCR006) cell dissociation solution for 20 minutes at RT under mild agitation. CUT&RUN experiments were carried out as described(Skene and Henikoff, 2017).
GEO Accession
GSM4120727