GSM8901745: WT zebrafish, biol rep 4, 5 days post-fertilization; Danio rerio; RNA-Seq
Total RNA was isolated using the NZYOL reagent. DNase treatment was then performed using the RNase-Free DNase set from QIAGEN. RNA samples were subjected to the RNeasy kit (QIAGEN) clean-up protocol. mRNA was purified from total RNA using poly-T oligo-attached magnetic beads and subjected to fragmentation. Then, the first strand cDNA was synthesized using random hexamer primers and M-MuLV Reverse Transcriptase, followed by the second strand cDNA synthesis using DNA polymerase I, RNase H and dUTP for directional library. The lasting overhangs were transformed into blunt ends through polymerase and exonuclease reactions. To prepare for hybridization, adaptors containing a hairpin loop structure were ligated to the previously 3' ends adenylated DNA fragments. The AMPure XP system was used to purify the cDNA fragments with 370~420 bp in length that were then digested with USER enzyme. PCR was carried out using Phusion High-Fidelity DNA polymerase, universal PCR primers and Index (X) primer. The PCR products were purified, and the Agilent Bioanalyzer 2100 system was used to evaluate the library quality.