GSM4259014: CTMB_rep2; Danio rerio; RNA-Seq
Brain and liver tissue isolation was performed by anaesthetizing the zebrafish by treatment with Tricaine (Sigma, USA). Utmost care was taken while dissecting out the tissue to ward off contamination to obtain a pure sample for each tissue type. Harvested tissues were immediately submerged in RNALater (~200 μl in total volume; Sigma) for storage in RNase-free microcentrifuge tubes (Ambion). Tissues were later stored at -80 °C. Total RNA was prepared using NucleoSpin® RNA kit (Macherey-Nagel, REF # 740955.50) as mentioned by the manufacturer's protocol. Tissues were separated from RNALater and were homogenized in the lysis buffer (from RNA kit) using a hand-held microtube homogenizer. On-column DNase digestion was performed to eliminate genomic DNA contamination.RNAs were eluted from the column using RNase and DNase free water (Sigma). After purification, the quantity and quality of RNA was determined using NanoDrop spectrophotometer and 1% RNA Agarose gel electrophoresis, respectively. All RNA samples were stored at -80 °C. TruSeq RNA Library Prep Kit (v2 LT) Non-stranded kit was used to prepare libraries for sequencing as per manufacturer's instructions.