title: GSM5509230: tRNA_Shield; Danio rerio; RNA-Seq
library_construction_protocol: About 50-100 zebrafish eggs or embryos at a desired stage were collected to extract total RNA, and dechorionated by Pronase digestion. 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. Total RNA was placed on ice for 30 min with 10% PEG8000 and 0.5 M NaCl, then centrifuged at 14,000 rpm for 15 min at 4℃. Supernatant (low molecular weight, LMW RNA) was collected and precipitated  by addition of ethanol. The LMW RNA was separated by 8% 8 M Urea PAGE gel, then the bands correspond to 60-120 nt length were cut and crunched into small pieces. RNA was eluted from the gel by 0.3 M NaOAc overnight on a vertical rotator at 4℃. Then the supernatant was precipitated at -20℃ after addition with 2 μl of Glycoblue and 2.5 volume of ethanol. After centrifuged at 12,000 rpm for 30 min and washed by 75% ethanol, the RNA precipitant was dissolved in RNase free water.  There are three pretreatment steps before library preparation. 60-120nt RNA enriched in the previous step was first incubated in 100 mM Tris-HCl pH 9.0 at 37℃ for 30 min to be deacylated, then the RNA was treated with T4 PNK to repair ends at 37℃ for 30 min. Modifications on tRNAs were removed by ALKB treatment [1 μg purified RNA was treated by ALKB protein mixture (wt ALKB, 160 pmol; ALKB D135S, 200 pmol; and ALKB D135S/L118V, 200 pmol) in reaction buffer (300 mM KCl, 2 mM MgCl2, 50 μM (NH4)2Fe(SO4)2, 300 μM α-ketoglutarate, 2 mM vitamin C, 50 μg/ml BSA, 50 mM MES pH 5.0, 40 U RNase Inhibitor, 1x Protease Inhibitor) for 2 h at room temperature]. Purification was needed after each pre-treatment step. 100 ng RNA was then subjected for RNA library preparation. tRNA library was prepared by VAHTS Small RNA Library Prep Kit for Illumina (Vazyme, NR801). RNA was heat denatured at 80℃ for 2 min in the first step, then every steps were done followed the manual in kit. The libraries were first concentrated by DNA concentrator spin column (Zymo), then separated on 6% native PAGE gel. Bands correspond to 180-240 bp (60-120 nt RNA ligated with both adaptors) length were cut and DNA libraries were eluted from gels.