title: Illumina HiSeq 2500 sequencing; RNA-seq transcriptomic analysis of mouse sympathetic neuroblasts and adrenal chromaffin cells
design: RNA-seq transcriptomic analysis of mouse sympathetic neuroblasts and adrenal chromaffin cells
library_construction_protocol: E12.5 TH-IRES-Cre;ROSA26-EYFP mouse embryos were generated by mating heterozygous TH-IRES-Cre mice with homozygous ROSA26-EYFP mice. The urogenital ridges were dissected by inserting the tips of fine forceps underneath, and pulled away by holding the dorsal aorta. The tissues were then dissociated and sorted by BD FACSAria Fusion cell sorter with a 100 µm nozzle at 20 psi. Live EYFP+ single cells were first isolated on the basis of 7-AAD (excitation: 488 nm, emission 692/40 nm) and then sorted into EYFP Hi and Lo. Total RNA from isolated EYFP+HI and EYFP+LO cells were extracted using by TRIzol/chloroform extraction followed by RNeasy Micro Kit (Qiagen). RNA pellets with ~20,000 cells from more than 10 embryos with a sex ratio within a range of 1:1.3 (male:female or female:male) were pooled and quantified that RIN> 8, average RIN = 9.7 cDNA library was prepared by random priming using the SMART-seq v4 Ultra Low Input RNA Kit (Clontech, Cat. 634889) followed by PCR amplification with the addition of Illumina adaptors for multiplexing experiment using Nextera XT Kit (Illumina, Cat. FC-131-1096)