GSM8945646: degradome_tas2-3_Rep2; Arabidopsis thaliana; RNA-Seq
Total RNA was extracted from 12-day-old Arabidopsis seedlings using the TRIzol method (Takara, 9108). sRNA-seq: Small RNAs (sRNAs) of varying lengths were separated by 15% Urea-PAGE, and the 15-40bp bands were excised post-electrophoresis. The gel slices were soaked overnight at 4℃ in 0.4M NaCl solution, followed by ethanol precipitation. RIP-seq: WT and mutant seedlings underwent treatment with cell lysis buffer (1M Tris-HCl, 5M NaCl, 10% NP-40, 1M MgCl2, 1M DTT, proteinase inhibitor cocktail (100x), RNase inhibitor) to extract total protein. Subsequently, AGO1 antibody (Agrisera, #AS09527) and Protein A dynabeads (Thermo, 10001D) were utilized to enrich AGO1 protein. Following this enrichment process, RNA extraction and construction of the small RNA (sRNA) sequencing library were conducted as previously described. mRNA-seq: Initially, polyA-tailed RNAs were selectively enriched from the total RNA pool using oligo d(T) beads. These enriched RNAs were subsequently fragmented into random segments. Utilizing these segments as templates and dNTPs as building blocks, double-stranded cDNA was synthesized. Following purification, end repair, and adapter ligation, cDNA segments measuring 370-420bp were selected for amplification to create the RNA sequencing library. degradome-seq: The comprehensive protocol for constructing the degradome sequencing library is elaborated in Li, Yong-Fang et al.