GSM3406903: iCLIP 4hpf rep1; Danio rerio; OTHER
RIP-seq: Briefly, 500 zebrafish embryos at shield stage (4 hpf) were resuspended with 2 ml lysis buffer (150 mM KCl, 10 mM HEPES pH 7.6, 2 mM EDTA, 0.5% NP-40, 0.5 mM DTT, 1:100 protease inhibitor cocktail, 0.4 U/μl RNasin) by rotating at 4°C for 30 min and centrifuged at 15,000 g for 15 min. The supernatant was collected and pre-cleared with protein A Dynabeads by rotation at 4°C for 1 h. 50 μl pre-cleared embryo lysate was saved as Input and the left were incubated with affinity purified rabbit polyclonal anti-Ybx1 antibody (prepared by AbMax, China) and 30 μl Protein A Dynabeads for 4 h at 4°C or overnight. After washing with1 ml ice-cold NT2 buffer (200 mM NaCl, 50 mM HEPES pH 7.6, 2 mM EDTA, 0.05% NP-40, 0.5 mM DTT, 0.4U/µl RNasin) for eight times and once with 1ml ice-cold 1× PK buffer (100 mM Tris-HCl pH 7.4, 50 mM NaCl, 10 mM EDTA , 0.2% SDS), the beads was treated in 200 ul PK buffer containing 20 ul proteinase K (Roche, 0311582001) for 1 h at 55°C. The solution was collected and subjected to RNA extraction with Acid Phenol: ChCl3 (pH4.3~4.7) and ethanol precipitation. The Input RNA was extracted by using TRIzol reagent. Both the Input and IP RNA were treated by TURBO™ DNase (Invitrogen, AM2238).  iCLIP-seq: 1000 zebrafish embryos at 4 hpf were irradiated twice with 0.8 J/cm2 (Stratalinker 2400, Stratagene), lysed and subjected to mild fragmentation. Crosslinked RNA-protein complexes were immunoprecipated using polyclonal Ybx1 antibody (Abmax) and protein A dynabeads. RNA was extracted and subjected to library construction using Smarter smRNA-Seq kit (Takara). All the libraries were sequenced using HiSeq2500 (Illumina) in paired-read mode, creating reads with a length of 150 bp.