GSM7899859: Nfur Control-KO Development RNAseq Rep9; Nothobranchius furzeri; RNA-Seq
For each stage in each species, roughly 8-30 embryos were carefully dissected in ice-cold PBS using biological-grade tweezers (Electron Microscopy Sciences, 72700-D) to carefully remove the chorion, the enveloping layer, and the yolk without damaging the embryo body. Freshly dissected embryos were then quickly rinsed with ice-cold PBS, and all the PBS was carefully removed. Embryo bodies were then snap-frozen in liquid nitrogen and stored at -80°C. We used 8-10 snap-frozen embryos for RNA-seq. Snap frozen embryos at -80°C were thawed on ice for 1 minute and washed with 200µl ice-cold PBS. The embryos were then dissociated and homogenized with ~25 Zirconia/Silicon 0.5mm glass beads (RPI, Research Products International Corp, 9834) using FastPrep® -24 homogenizer (MB Biomedicals, 116004500) for 20 seconds, followed by centrifugation (17000g for 3 minutes). After centrifugation, 10.5µl of the supernatant was used as input to the SMART-Seq® v4 Ultra® Low Input RNA Kit (Takara, 634890) for the cDNA synthesis followed by amplification with 12 cDNA amplification cycles. Amplified cDNA was validated with Agilent 2100 Bioanalyzer using Agilent's High Sensitivity DNA Kit (Agilent, Cat. No. 5067-4626). The DNA libraries were then generated using the Nextera XT DNA Library Prep Kit (Illumina, FC-131-1096). Library quality and concentration were assessed by the Agilent 2100 Bioanalyzer and Agilent's High Sensitivity DNA Kit (Agilent Technologies, Cat. No. 5067-4626), followed by high throughput sequencing on Illumina HiSeq platform with 2 x 150bp paired end reads.