GSM5709366: Eye opn4 dko biol rep 3; Danio rerio; RNA-Seq
Eyes were removed by cutting the optic nerves with surgical scissors and the brain was carefully removed after lifting the skullcap with surgical forceps and cutting the spinal cord. The brain was separated in an anterior and posterior part with a surgical blade. For each biological replicate the same tissue parts of three individuals were pooled and frozen in liquid nitrogen. A total of three biological replicates were processed. Total RNA was isolated by adding RNAzol (Sigma-Aldrich) and a steal bead to the frozen sample, followed by disruption in a bead mill for 3 min at 30 Hz (TissueLyser II, Qiagen) and extraction of total RNA (Direct-zol RNA kit, Zymo Research). mRNA was isolated from total RNA by selective binding to oligo-dT coupled beads (Dynabeads mRNA purification kit, Invitrogen) followed by washes and elution. To increase purity the eluate was bound, washed, and eluted for a second round, and thereafter quality checked (Bioanalyzer RNA 6000 Pico assay, Agilent). Next, mRNA was fragmented for 3 min at 75 °C (fragmentation reagents, Invitrogen) and the fragment size distribution was quality checked (Bioanalyzer RNA 6000 Pico assay, Agilent). mRNA was transcribed with the SuperScript VILO cDNA synthesis Kit (Invitrogen) and libraries were constructed with the ultra II kit (NEB), followed by adding barcodes with PCR (multiplexed). The samples were single read 100bp RNA sequenced with the Illumina HiSeq 2500 v4 chemistry.