GSM2845159: RNAseq_Epiboly_Negatives1; Danio rerio; RNA-Seq
FACS sorted cells were washed with PBS and stored at -80C in lysis buffer. RNA was extracted using Ambion RNAqueous Micro Total RNA isolation kit (AM1931), checked on Bioanalyser, samples with RIN >7 were used to prepare cDNA using Takara Clontech SmartSeq2 V4 kit (634889). Sequencing libraries were prepared using Illumina Nextera XT library preparation kit (FC-131-1024). RNA pools extracted using RNAqueous Micro Scale Total RNA Isolation Kit. Non-directional sequencing libraries after polyA-selection of RNA transcripts (NEBNext® Poly(A) mRNA Magnetic Isolation Module, NEB) were built using NEBnext Ultra RNA library kit for Illumina (NEB). Following ribodepletion using Ribo-ZeroTM Magnetic Kit (Epicentre), directional RNA-seq libraries were constructed using Stranded RNA-Seq Library Preparation Kit (KAPABiosystems).