GSM5179370: ribosomes 4h WT rep2; Danio rerio; RNA-Seq
Generally, Zebrafish embryos were incubated in 100 mg/ml cycloheximide in E3 buffer (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, 0.33 mM MgSO4) for 5 min at room temperature. Then embryos were then transferred into 600 ul of ice-cold lysis buffer. Clarify the lysate by centrifugation for 10 min at 20,000g after Triturate the cells ten times through a 26-G needle, and recover the soluble supernatant. Add 6 ul of RNase I (100 U µl−1) to 600ul lysate. Incubate for 45 min at room temperature with gentle mixing. Add 10.0 µl of SUPERase·In (Life Technologies, AM2694) RNase inhibitor to stop nuclease digestion. Meanwhile, MicroSpin S-400 HR columns (GE Healthcare, 27-5140-01) were equilibrated with 3 ml of mammalian polysome buffer by gravity flow and emptied by centrifugation at 600g for 4 min. We then immediately loaded 100 µl of the digested lysate on the column and eluted the column by centrifugation at 600g for 2 min. We extracted RNA from the flow-through (approximately 125 µl) using Trizol LS (Life Technologies, 10296-010). We  removed ribosomal RNA fragments using the RiboZero Kit (Illumina, MRZH11124) and separated them on a 17% denaturing urea-PAGE gel (National Diagnostics, EC-829). The size range from 27 nt to 30 nt was cut out, and the RNA fragments were subjected to library generation using Smarter smRNA-Seq kit (Clontech Laboratories Inc).
GEO
GSM5179370