GSM9687354: Smox MO MTZ 3; Danio rerio; RNA-Seq
pool 20-50 zebrafish embryos in a 1.5 ml microfuge tube and remove as much water as possible with a pipette. Immediately add 250 ul of IRizol reagent' to the microfuge tube containing the embryos. TRizol reagent contains phenol and should only be used under a fume hood. Lyse and homogenize the embryos with a pellet pestle (approximately 20 strokes) until the tissue is sufficiently disrupted. When the cells are sufficiently homogenized, add 750 ul TRIzol reagent to equal a total volume of 1 ml. To permit complete dissociation of nucleoprotein complexes, incubate homogenized samples for 5 minutes at room temperature. At this stage, the sample can be flash frozen in liquid nitrogen and stored at -80°C or the protocol can be continued. To continue add 0.2 ml of chloroform and rock the tube for 15 seconds to mix. Incubate the sample for 2 minutes at room temperature and then centrifuge at 12,000 x g for 15 minutes at 4°C. The mixture will separate into a lower red phenol-chloroform phase, an interphase, and a colorless upper aqueous phase. The upper aqueous phase contains the RNA. The top layer should comprise of about 60% of the initial volume of TRIzol (approximately 0.6 ml). Transfer the top aqueous layer into a new microfuge tube using a 1 ml pipette being careful not to transfer any of the interphase layer. To precipitate the RNA add 0.5 ml isopropanol. Allow the sample to sit at room temperature for 10 minutes and then centrifuge the sample at 12,000 x g for 10 minutes at 4°C. RNA will form a gel-like pellet on the bottom of the tube. Without disturbing the pellet, remove the supernatant with a pipette and wash the pellet in 1 ml 75% ethanol. Mix the sample by gentle inversion and centrifuge at 7,500 x g for 5 minutes at 4°C. After centrifugation, remove the ethanol with a pipette and allow sample to dry. Resuspend the pellet by adding 100 ul of RNAse-free water and incubate the sample at 55°C for 10 minutes. mRNA enrichment was performed on total RNA using oligo(dT)-attached magnetic beads. The enriched mRNA with poly(A) tails was fragmented using a fragmentation buffer, followed by reverse transcription using random N6 primers to synthesize cDNA double strands. The synthesized double-stranded DNA was then end-repaired and 5'-phosphorylated, with a protruding 'A' at the 3' end forming a blunt end, followed by ligation of a bubble-shaped adapter with a protruding 'T' at the 3' end. The ligation products were PCR amplified using specific primers. The PCR products were denatured to single strands, and then single-stranded circular DNA libraries were generated using a bridged primer. The constructed libraries were quality-checked and sequenced after passing the quality control.