GSM6300337: zf_library_8h_1; Danio rerio; OTHER
Injected zebrafish embryos were collected in Buffer RLT (QIAGEN RNeasy kit), followed by vortexing for 1 min and stored at -70°C until extraction. RNA was thawed at 37°C, extracted following the manufacturer's protocol, and eluted in 50 μL. mRNA was isolated with Dynabeads oligo-(dT) mRNA purification kit following the manufacturer's protocol. mRNA was eluted in 10 μl of 10 mM Tris-HCl, pH 7.5. 5 μL of purified mRNA were used for cDNA synthesis using the SuperScript IV Reverse Transcriptase kit, utilizing a specific oligo that binds to the 3' Illumina adapter (GTAGTGACTGGAGTTCAGAC). The cDNA was cleaned with the Qiagen MinElute PCR purification kit and eluted in 20 μL of water. We tested different PCR cycles to amplify the library using specific oligos that target the surrounding Illumina adapters. We selected 16 cycles for the samples at 2 and 5 hpi, and 18 cycles for 8 hpi. The barcoded libraries were cleaned using AMPure XP beads and resuspended in 20 μL of buffer EB (Qiagen). The libraries were sequenced in an Illumina MiSeq instrument 250bp pair-end with 10% of PhiX as spike-in. 