GSM9056081: Seedling, Mock, Ribo-seq rep 3; Arabidopsis thaliana; OTHER
For RNA-seq, RNA is extracted using TRIZOL reagent. For Ribo-seq, plant samples are lysed using an appropriate method. After nucleic acid digestion, ribosome-bound RNA is separated from free mRNA. Ribosome-mRNA complexes are then enriched to obtain ribosome-protected fragments (RPFs). For RNA-seq, the enriched mRNA is subjected to fragmentation, and the RNA fragments are reverse-transcribed into cDNA. Sequencing adapters are added to the cDNA. PCR amplification is carried out on the adapter-containing cDNA fragments to enrich for those with specific adapters, thereby forming the final sequencing library. For Ribo-seq, Lyse plant samples, nucleic acids undergo digestion to separate ribosome-bound RNA from free mRNA. Ribosome-mRNA complexes are then enriched to obtain ribosome-protected fragments (RPFs). Following the removal of rRNA and purification by PAGE gel electrophoresis, the target RNA fragments are acquired. The ends of these fragments are repaired, and 5' and 3' adapters are directly added. Reverse transcription is carried out to synthesize cDNA, which is subsequently purified using PAGE gel. Sequencing adapters are attached, and PCR amplification is performed to enrich the fragments, resulting in the initial library. Finally, PAGE gel is used to screen for the target fragments, yielding a library containing the desired fragments.