GSM4979509: TU + control morpholino + DMSO, tailbud stage, replicate #3; Danio rerio; RNA-Seq
109 snap-frozen embryos of each genotype and treatment group (in triplicate) were homogenized in 700 µl TRIzol (Invitrogen/Thermo Fisher Scientific, 15596026) using a tissue homogenizer with hard tissue Omni tip plastic homogenizing probes (Omni International, 30750H). 300 µl TRIzol was added and the samples transferred into a pre-spun (16,000 x g, 30 s, room temperature) 2-ml 5PRIME Phase Lock Gel Heavy tube (Quantabio, 2302830). After addition of 200 µl chloroform (Sigma-Aldrich, C2432), the tube was vigorously inverted for 15 sec, incubated at room temperature for 3 min and spun at 12,000 x g for 15 min at 4°C. The supernatant was transferred into a RNase-free 1.7-ml tube. 300 µl buffer-saturated phenol (Invitrogen/Thermo Fisher Scientific, 15513039) and 300 µl chloroform were added, the samples vortexed for 15 sec, and incubated and spun as before. The supernatant was transferred into a fresh RNase-free 1.7-ml tube, 600 µl chloroform added, the samples were vortexed, incubated and spun as before. The supernatant was transferred into a fresh RNase-free 1.7-ml tube, 600 µl 2-propanol (VWR, BDH1133) was added, the samples were inverted 10 times and stored at -80°C overnight. Samples were spun at 17,000 x g for 30 min at 4°C, and the supernatant was pipetted off. Samples were handled on ice from now on. Samples were washed three times with 1000 µl 75% ethanol in nuclease-free water (Invitrogen/Thermo Fisher Scientific, AM9937) followed by spinning at 17,000 x g for 5 min at 4°C. After the final wash, samples were air-dried and resuspended in 50 µl nuclease-free water. 5 µg of RNA was DNase-treated with the TURBO DNA-free Kit (Ambion, AM1907) as per the manufacturer's protocol, in a volume of 50 µl for 30 min at 37°C. Instead of adding the DNase Inactivation Reagent, the reaction was cleaned up using RNAClean XP SPRI paramagnetic beads (Beckman Coulter, A63987) following the manufacturer's instructions. The final elution volume was 48 µl. 1.5 µg DNase-treated RNA samples were spiked with ERCC ExFold RNA Spike-In Mixes (Thermo Fisher Scientific, 4456739) as per the manufacturer's protocol. Ribosomal RNAs were removed with the Ribo-Zero Magnetic Gold Kit (Illumina, MRZG12324), and strand-specific libraries were prepared with the NEBNext Ultra Directional RNA Library Prep Kit for Illumina (NEB, E7420) and NEBNext Multiplex Oligos for Illumina (NEB, E7335) all according to the manufacturer's protocols. For quality control and pooling purposes, the final libraries were run on a high sensitivity DNA chip (Agilent, 5067-46426) on the Agilent 2100 Bioanalyzer. The average library size was 265 bp. Libraries were sequenced on the Illumina HiSeq 4000.