syn2syn4-RNAseq-biological replicate3
The total RNAs were extracted from 7-day-old Arabidopsis seedlings using Trizol reagent (TIANGEN)After the RNA samples were qualified, the magnetic beads with oligo (DT) were used to enrich the eukaryotic mRNA (in the case of prokaryotes, the rRNA was removed by the kit to enrich the mRNA). After that, the fragment buffer was added to break the mRNA into short fragments. Using the mRNA as the template, random hexamers were used to synthesize a strand of cDNA. Then the buffer solution, dNTPs, DNA polymerase I and RNase H were added to synthesize the two strand cDNA. Then the double strand cDNA was purified by ampere XP beads. The purified double stranded cDNA was repaired at the end, added a tail and connected to the sequencing connector, and then ampure XP beads was used to select the fragment size. Finally, the PCR products were amplified by PCR and purified by ampure XP beads to obtain the final library. After the library was constructed, qubit 2.0 was used for preliminary quantification, and the library was diluted. Then, Agilent 2100 was used to detect the insert size of the library. After the insert met the expectation, Q-PCR method was used to accurately quantify the effective concentration of the library to ensure the quality of the library.