Ribo-seq of csp2 under 22 degrees Celsius
Young flowers of 35-DAG csp2 mutant under 22 degrees Celsius for 12 h were collected and then ground in liquid nitrogen followed by resuspension in 600 L ice-cold lysis buffer. 6 L of RNase I (100 U/L) were added to 600 L lysate to separate ribosome-bound RNA from free mRNA. This process was designed to enrich the ribosome-mRNA complex, thereby obtaining ribosome-protected fragments (RPFs). Subsequently, RNA fragments around 30 nucleotides in length were purified and recovered using PAGE (Polyacrylamide Gel Electrophoresis) gel. Following recovery, the RNA samples were subjected to end-repair procedures, followed by the addition of adapters at both ends.