RNA-seq of Xenopus laevis; whole eye isolated at NF st. 24
RNA samples were collected from Xenopus laevis embryos at three district developmental stages: 24, 27, and 35. Each stage was collected in triplicates (n = 3) and each biological replicate was composed of pooled eye tissue from 20-30 animals/embryos. Eye ablation and tissue dissociation was performed in Liberase (Thermolysin Medium Research Grade, Roche, Indianapolis, IN) and/or calcium-magnesium free Modified Ringer solution (CMF-MR). Dissociated tissues were immediately stored in TRIzol Reagent (Invitrogen, Carlsbad, CA) at 80C. Total RNA was isolated using the Direct-zol RNA Microprep Kit (Zymo Research) according to the manufacturers instructions. RNA-seq libraries were prepared from the extracted RNA using the Illumina TruSeq Stranded mRNA Library Prep Kit, utilizing 0.8 to 3.2 g of input RNA. A diluted "A-Tailing Control" was introduced during ligation. Library quality was assessed using Agilent Bioanalyzer and molarity of the libraries was quantified using Roche KAPA qPCR. Paired-end sequencing with 150 cycles was performed on an Illumina NextSeq500 platform, and raw sequencing data were obtained from BaseSpace.