GSM5069283: Sphere-ncRNA-seq; Danio rerio; ncRNA-Seq
For small RNA sequencing: About 50-100 zebrafish eggs or embryos at a desired stage were dechorionated by Pronase digestion and collected to extract total RNA. Embryos were then transferred to 1.5-ml eppendorf tube, and lysed in 1 ml Trizol Reagent (Thermo Fisher Scientific, 15596018). The lysate was centrifuged at 12,000 rpm for 10 min, and the supernatant was collected and mixed with 200 μl chloroform in a fresh tube. After centrifugation at 12,000 rpm for 10 min at 4℃, the supernatant was transferred to a fresh tube with addition of equal volume of isopropanol and incubated at room temperature for 15 min, followed by thorough mix and centrifugation at 12,000 rpm for 10-20 min. The RNA pellet was washed by 75% ethanol once and was stored as pellet in 75% ethanol at -80℃ until all samples were collected. Two pretreatment steps were included: First, purified total RNAs were treated with T4 Polynucleotide Kinase (NEB, M0201S) with ATP for 30 min at 37℃, then purified by phenol: chloroform extraction. The aim of this step is removing the 2,3'-cyclic phosphate group from the 3'end of 5'tRFls, and add phosphate group to the 5'end of 3'tRFs. In the second step, total RNAs were treated with ALKB protein mix as mentioned above. Purified total RNAs were then subjected to small RNA library preparation. For small RNA sequencing:  Small RNA libraries, using 1 μg RNA each, were prepared using the NEBNext Multiplex Small RNA Library Prep Set for Illumina (NEB) according to the manual. The amplified DNA from each sample was first concentrated by DNA concentrator spin column (Zymo Research), then bands of 135-160 bp, about the length of 15-40 nt RNA ligated with both adaptors, were manually selected on native PAGE gel. DNAs were eluted from the gel piece with 0.3 M NaOAc, pH 4.5, followed by precipitation by adding 2.5 volumes of ethanol.