sRNA seq of A. thaliana plants transformed with pYAO-Cas9-U6-MIR390a-sgRNA2-dFT
Steps of library preparation by BGI: 1) Small RNA selection: Use PAGE electrophoresis to cut and separate RNA of size 18-30nt from total RNA; 2) 3' adapter ligation: Use 5-adenylated and 3-blocked single stranded DNA adapter to ligate with the 3' end of RNA from step (1); 3) Annealing of reverse transcription primers: Add RT primers into the system from step (2), hybridize with the 3' adapter attached to the RNA, meanwhile hybridize with excess 3' adapter; 4) 5' adapter ligation: Ligate the 5' adapter to the 5' end of products from step (3). Since the adapter preferentially ligates with single-stranded molecules rather than the hybrid of 3' adapter and RT primer, it can greatly reduce the ligation between adapters; 5) Synthesize the first strand of cDNA: Use RT primer from step (3) for reverse transcription extension, then synthesize the first strand of cDNA; 6) PCR amplification: Use high-sensitivity polymerase to amplify cDNA, enrich the cDNA with both 3' adapter and 5' adapter, then amplify the library yield; 7) Library fragment selection: Use PAGE electrophoresis to separate PCR products in the range of 100-120bp, which effectively removes by-products such as primer dimers; 8) Library quantification and pooling cyclization