GSM8245570: zebrafish embryo,22C,1hpf,1; Danio rerio; RNA-Seq
Total RNA was isolated using tri-reagent (Sigma T9424) and quantified using nanodrop We used a two-step RNA-Seq library construction approach that specifically targeted the variable 3′UTR region of reporters. In the first step, total-RNA was reverse-transcribed with Maxima RT (Thermo Fisher) and a gene-specific primer that matched the 20nt downstream 3′UTR universal adaptor. RT primer also added a random 8nt UMI and a constant RT adaptor sequence. In the second step, resulting cDNA was amplified by 18 cycles of Phusion PCR (NEB) with primers that matched the 20nt upstream 3′UTR universal adaptor and the RT adaptor. PCR primers also added the appropriate Illumina sample barcodes and sequencing adaptors,  resulting in the final RNA-Seq library.