GSM4476139: Crushed optic nerves, replicate 3 from hibernators; Ictidomys tridecemlineatus; RNA-Seq
Enucleated eyes were dissected to form posterior segment eye cups, which were then fixed in 4% paraformaldehyde in phosphate buffered saline(PBS) for 2–4 h at 4°C. Eyecups were either processed for vibratome sectioning (100um-thick sections, VT1000; Leica) or dissected to form retinal flat mounts. Flat-mounted retinas or retinal sections were blocked overnight in blocking buffer containing 6% normal donkey serum and 0.5% TritonX-100 in PBS at room temperature. GS retinas and optic nerve tissue were collected and lysed in TRIzol (Thermo Fisher, Waltham, MA), total RNA was isolated and purified with miRNeasy kit (Qiagen, Hilden, Germany). TruSeq Stranded mRNA Library Prep Kit was used to generate the sequencing libraries (Illumina, San Diego, CA) following the manufacturer's instructions.