GSM8579723: Zebrafish, RNAseq, M-24HPF-1; Danio rerio; RNA-Seq
Total RNAs were extracted from WT and Mrbm24a embryos at 4-cell sphere 24-hpf stage, using TRIzol reagent (Invitrogen), and were precipitated by cold isopropanol (50%, v/v) in the presence of carrier glycogen (20 µg/each sample). 1 μg total RNA was used for following library preparation. The poly(A) mRNA isolation was performed using Oligo(dT) beads. The mRNA fragmentation was performed using divalent cations and high temperature. Priming was performed using Random Primers. First strand cDNA and the second-strand cDNA were synthesized. The purified double-stranded cDNA was then treated to repair both ends and add a dA-tailing in one reaction, followed by a T-A ligation to add adaptors to both ends. Size selection of Adaptor-ligated DNA was then performed using DNA Clean Beads. Each sample was then amplified by PCR using P5 and P7 primers and the PCR products were validated. Then libraries with different indexs were multiplexed and loaded on an Illumina HiSeq/ Illumina Novaseq/ MGI2000 instrument for sequencing using a 2x150 paired-end (PE) configuration according to manufacturer's instructions.