GSM8759066: Zebrafish, RIP-seq, Rbm24a Knock-in1 IgG1; Danio rerio; RIP-Seq
Total RNAs were extracted from 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 of immunoprecipitated RNA was utilized for library construction. Ribosomal RNA depletion was performed using RNase H-based probes followed by magnetic bead purification. RNA fragmentation was achieved through magnesium-catalyzed hydrolysis under elevated temperature (94°C for 6 min). First-strand cDNA synthesis was initiated with random hexamer primers and reverse transcriptase, followed by second-strand synthesis using DNA polymerase I with dUTP incorporation for strand marking. Double-stranded cDNA fragments underwent end repair and 3'-adenylation using a single-tube enzymatic reaction system. Illumina-compatible adaptors with unique dual indexes were ligated to both ends of the DNA fragments. Size selection of adaptor-ligated products (180-250 bp) was performed using double-sided SPRI bead purification. PCR amplification was conducted with P5/P7 primers for 12 cycles, followed by AMPure XP bead purification to remove primer dimers. Library quality was verified by Agilent 2100 Bioanalyzer DNA High Sensitivity chip analysis. Indexed libraries were pooled in equimolar ratios and subjected to paired-end sequencing (2×150 bp) on an Illumina NovaSeq 6000 platform following the manufacturer's standardized workflow.