GSM1357159: cntl_neo_1hr_1; Danio rerio; RNA-Seq
Two hundred 5dpf untreated Tg(sqET20) control or neomycin treated Tg(sqET20) larvae were anesthetized, collected in a 2mL tube, dissociated with trypsin and filtered to remove un-dissociated tissue. A two-gate FACS strategy was used to select only living target cells from excesses of dead cells and cellular debris of the larval cell suspensions (see Materials and Methods of associated manuscript for detailed FACS protocol). Approximately 30,000 GFP- or GFP+ cells were used for total RNA extraction using Trizol (Invitrogen) following the manufacturer’s manual. Libraries for RNA sequencing were made with poly-A selected mRNA using the Illumina TruSeq RNA library construction kit v2 (Illumina). The resulting libraries were purified using Agencourt AMPure XP system (Backman Coulter), then quantified using a Bioanalyzer or Qubit Fluorometer (Life Technologies).