Rnaplant regent

RNA ligase-mediated rapid amplification of 5′cDNA ends (RLM-RACE) GeneRacer Kit (Invitrogen, USA) was used to validate miRNA-guided mRNA cleavage, which differed with traditional 5′RACE of full-length cDNA by omitting the 5′ phosphates of truncated mRNA removal and the 5′ cap structure of full-length mRNA removal treatments. Briefly, total RNA was extracted with RNAplant regent (TIANGEN, DP407-01), and PolyA RNA was isolated using polyAtract mRNA isolation system III (Promega, USA) to eliminate contaminated non-mRNA. Ligation with a 5′ RNA adapter and a reverse transcription were performed. The resulting cDNA was used as a template for PCR amplification. Two ~100 bp spaced gene specific reverse primers (GSP1 and GSP2) for each target, designed based on the downstream sequence of the miRNA:target binding site at the target gene sequence. Combining with two GeneRacer 5′ forward primers (included in GeneRacer kit) to specifically nest amplify the 3′ cleavage product of the target mRNA. The amplified PCR products were gel purified, cloned and sequenced (Sangon, China). Gene specific primers that we used are provided in Additional file 5: Table S7.

RNA ligase-mediated rapid amplification of 5′cDNA ends (RLM-RACE) GeneRacer Kit (Invitrogen, USA) was used to validate miRNA-guided mRNA cleavage, which differed with traditional 5′RACE of full-length cDNA by omitting the 5′ phosphates of truncated mRNA removal and the 5′ cap structure of full-length mRNA removal treatments. Briefly, total RNA was extracted with RNAplant regent (TIANGEN, DP407-01), and Poly(A) RNA was isolated using polyAtract mRNA isolation system III (Promega, USA) to eliminate contaminated non-mRNA. Ligation with a 5′ RNA adapter and a reverse transcription were performed. The resulting cDNA was used as a template for PCR amplification. Two ~100 bp spaced gene specific reverse primers (GSP1 and GSP2) for each target, designed based on the downstream sequence of the miRNA:target binding site at the target gene sequence. Combining with two GeneRacer 5′ forward primers (included in GeneRacer kit) to specifically nest amplify the 3′ cleavage product of the target mRNA. The amplified PCR products were gel purified, cloned, sequenced (Sangon, China), and the cleavage site determined by align sequenced data to target gene. The target prediction of phased and half-phased miRNA-like RNAs is listed in Table S3, and gene specific primers that we used are provided in Table S4.

RNA ligase-mediated rapid amplification of 5′cDNA ends (RLM-RACE) GeneRacer Kit (Invitrogen, USA) was used to validate miRNA-guided mRNA cleavage, which differed with traditional 5′RACE of full-length cDNA by omitting the 5′ phosphates of truncated mRNA removal and the 5′ cap structure of full-length mRNA removal treatments. Briefly, total RNA was extracted with RNAplant regent (TIANGEN, DP407-01), and PolyA RNA was isolated using polyAtract mRNA isolation system III (Promega, USA) to eliminate contaminated non-mRNA. Ligation with a 5′ RNA adapter and a reverse transcription were performed then after. The resulting cDNA was used as a template for PCR amplification. Two ~100 bp-spaced gene-specific reverse primers (GSP1 and GSP2) for each target were designed based on the downstream sequence of the miRNA target binding site at the target gene sequence, and combined with two GeneRacer 5′ forward primers (included in GeneRacer kit) to specifically nest amplify the 3′ cleavage product of the target mRNA. The amplified PCR products were gel purified, cloned and sequenced (Sangon, China). Gene specific primers that we used are provided in Additional file 2: Table S14.