GSM4979516: mon clutch 3 at 30 hpf; Danio rerio; RNA-Seq
After thawing the samples, each with 30 embryos per phenotype and time point (in triplicate) homogenized in 500 µl TRIzol (Invitrogen/Thermo Fisher Scientific, 15596026), samples were 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 100 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. 150 µl buffer-saturated phenol (Invitrogen/Thermo Fisher Scientific, 15513039) and 150 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, 300 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, 250 µl 2-propanol (VWR, BDH1133) added, the samples were inverted 10 times, samples were spun at 17,000 x g for 10 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 28 µl. 1 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 NEBNext rRNA Depletion Kit v2 (NEB, E7400), followed by a clean-up with RNAClean XP SPRI paramagnetic beads (Beckman Coulter, A63987). Strand-specific libraries were prepared with the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB E7760) 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 HS D1000 ScreenTape in the Agilent 2200 TapeStation System. The average library size was 278 bp. Libraries were sequenced on the Illumina HiSeq 4000.