Polyatract mrna isolation system 3

The 5′ RLM-RACE (RNA ligase-mediated 5′ rapid amplification of cDNA ends) method was used for target validation (Park and Shin, 2014 (link)). The total RNAs of pond-treated samples were isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, United States) following manufacturer’s instructions. mRNAs were separated from the total RNA using PolyA Tract mRNA Isolation System III (Promega, United States), and were then used to perform 5′ RLM-RACE using GeneRacer^TM Kit (Invitrogen, Carlsbad, CA, United States), according to the instructions of the manufacturer. Gene-specific nested reverse primers (listed in Supplementary Table S1) were used in combination with the 5′ primer provided by the kit to amplify the cleaved transcripts. Amplicons were purified using the AxyPrep DNA gel extraction kit (Axygen, United States), cloned into the pEASY-T3 vector (Transgene, Beijing, China), and sequenced.

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.

The apicomplexan alga C. velia (strain CCAP 1602/1) was obtained from the Scottish Culture Collection of Algae and Protozoa (CCAP) and cultivated in 400 ml L1-medium at 22 °C. The respective 1-l Erlenmeyer flask was shaken with 100 rpm in the New Brunswick Scientific Innova 42 incubator shaker under continuous light. Eight hundred and 2,000 mg of algal material was pesteled in liquid nitrogen for DNA and total RNA isolation. One hundred fifty micrograms of genomic DNA was purified with the DNeasy Plant Mini Kit (Qiagen) and 240 µg of total RNA was isolated with the TRIzol reagent (Gibco BRL). The PolyATract mRNA Isolation System III (Promega) was used for the isolation of 300 ng mRNA.
The axenic dinoflagellate Prorocentrum minimum (strain CCMP 1329) was obtained from the Provasoli-Guillard National Center for Marine Algae and Microbiota and cultured without shaking in L1-Si medium at 22 °C. 110 nanograms mRNA was isolated from 900 mg of algal material.

The petals and sepals were collected from full bloom flowers of VMP and pooled as one tissue source (designated as petal-sepal). The adaxial and abaxial layers of both tissues were separated equally using a scalpel blade, and immediately placed on ice to prevent any RNA degradation. Two grams of each pooled adaxial and abaxial layers were subjected to total RNA extraction as described by [13 (link)]. PolyA + mRNAs were isolated from total RNA of each adaxial and abaxial layer, separately using the PolyATract^® mRNA Isolation System III (Promega, USA) following the manufacturer’s instructions. A total amount of 50 ng of polyA + mRNA of each pooled adaxial and abaxial layers was subjected to transcriptome library construction using the ScriptSeq™ v2 RNA-Seq kit (Epicentre, a Illumina company, USA). The transcriptome libraries were sent to Science Vision Sdn. Bhd. (Selangor, Malaysia) for sequencing using the MiSeq Desktop Sequencer (Illumina, USA).

To verify the nature of potential miRNA targets and to examine how the miRNAs regulate their target genes, an RNA ligase-mediated 5′ rapid amplification of cDNA ends (RLM-5′-RACE) experiment was set up. RLM-5′-RACE was carried out following the instructions of the GeneRacer Kit (Invitrogen), as described by Yin et al.38 (link). Briefly, poly(A)⁺ mRNA was isolated from total RNA using the polyAtract mRNA isolation system III (Promega, Madison, WI). The GeneRacer RNA Oligo adapter was directly ligated to poly(A)⁺ mRNA without calf intestinal phosphatase and tobacco acid pyrophosphatase treatment, according to the manufacturer’s instructions. The GeneRacer OligodT primer was then used to synthesize RACE-ready first-strand cDNA in a reverse transcription reaction. First-round PCR was performed using the GeneRacer 5′ primer and a gene-specific primer, followed by second-round PCR with the GeneRacer 5′ Nested Primer and a gene-specific nested primer, which was designed using Primer 5.0 software and was located 300–800 bp downstream from the potential cleavage sites. The gene-specific and nested primers used in these assays are listed in Supplementary Table 3. The nested PCR products were gel-purified, T-cloned and sequenced.