RNA-seq of lung: adult femal Protopterus amphibious
A female P. amphibius was immediately anesthetized by freezing, and the heart and lung tissues were rapidly clipped, snap-frozen in liquid nitrogen and stored at -80 prior to the RNA extraction. The total RNA of each tissue was extracted using a standard Trizol Reagent Kit following the manufacturers protocol, respectively. After the quantitative evaluation of total RNA was completed using the Agilent 2100 Bioanalyzer, we purified mRNA by depleting rRNA from total RNA using the RNA Purification Beads. The purified mRNA was cut into fragments of appropriate size, and fragmented mRNA was further used to construct the paired-end cDNA library. Paired-end cDNA libraries were generated following manufacturers recommendations and index codes were added to attribute sequences to each tissue. Subsequently, we diluted two paired-end cDNA libraries and the quality of two libraries were assessed on the Agilent Bioanalyzer 2100 system. The clustering of the index-coded tissues was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS according to the manufacturers instructions. After cluster generation, two tagged paired-end cDNA libraries were further sequenced on the Illumina HiSeq 2500 platform and 150bp paired-end reads were generated.