Combined 2
Firstly, ribosomal RNA was removed from total RNA,folowed by ethanoprecipitation. After fragmentation, the first strand cDNA was synthesizedusing random hexamer primers. During the second strand cDNAsynthesis, dUTPs were replaced with dTTPs in the reaction buffer. Thedirectional library was ready after end repair, A-tailing, adapter ligation,size selection, USER enzyme digestion, amplification, and purification.The library was checked with Qubit and real-time PCR for quantificationand bioanalyzer for size distribution detection. After the library QC, it is pooled according to the effective concentrationand the target data output required for the experiment, then subjected tolllumina sequencing, which produces 150 bp paired-end reads. The basicprinciple of the sequencing is "Sequencing by Synthesis," wherefluorescently labeled dNTPs, DNA polymerase, and adapter primers areadded to the sequencing flow cell for amplification. As each sequencingcluster extends its complementary strand, the addition of eachfluorescently labeled dNTP releases a corresponding fluorescence signal.The sequencer captures these fluorescence signals and converts theminto sequencing peaks through computer software, thereby obtaining thesequence information of the target fragment.