title: GSM8714879: RFP_RB70_rep3; Nicotiana tabacum; OTHER
library_construction_protocol: For Ribo-Seq, the ribosome footprints were generated using 250 U RNase I per 1 ml of plant lysate (derived from 100 mg of plant fresh weight). The lysate was then layered on a sucrose cushion to purify monosomes. Ribosome footprints from ~20 nt to ~39 nt were size-selected from polyacrylamide gels. For total RNA samples, RNA was isolated with the TRIzol® Reagent (Thermo Fisher Scientific) according to manufacturer's instructions, gDNA was removed using TURBO™ DNase. For Ribo-seq, cDNA was synthesized by using NEXTflex™ Small RNA-Seq Kit v3. For RNA-seq, cDNA libraries were generated using Zymo-Seq RiboFree total RNA library kit. The quality and quantity of cDNA libraries was measured by Bioanalyzer and Qubit, respectively.