Quick rna isolation kit

Total RNAs from the sixth leaves of wild-type and EsMYB90 transgenic tobacco at the 7–8 leaves stage were isolated using a Quick RNA isolation kit (Bioteke Corporation, Beijing, China). The RNA library construction and sequencing were performed in the BGI Corporation (Shenzhen, China) using the BGISEQ-500 platform. Three independent biological replicates were carried out.
The low-quality reads (more than 20% of the bases qualities are lower than 10), reads with adaptors and reads with unknown bases (N bases more than 5%) were filtered to get the clean reads. The clean reads were mapped to the reference genome using HISAT [65 (link)]. Meanwhile, the clean reads were mapped to the reference transcripts using Bowtie2 [66 (link)]. The clean reads were assembled into unigenes, followed by the unigene functional annotation, etc., and calculate the unigene expression levels of each sample [67 (link)]. Finally, we identified DEGs (differential expressed genes) and performed clustering analysis and functional annotations. DEGs with the GO and KEGG annotation results were classified according to the official classification, and the GO and KEGG pathway functional enrichment were performed using phyper in the R software. Transcription Factor Prediction of DEG: The ORF of each DEG were founded using getorf and aligned to TF domains (from PlntfDB) using hmmsearch [68 (link)].

Total RNAs from the sixth leaves of wild type and EsMYB90 transgenic tobacco at the 7–8 leaf stage were isolated using a Quick RNA isolation kit (Bioteke Corporation, Beijing, China). The RNA library construction and sequencing were performed in the BGI Corporation (Shenzhen, China) using the BGISEQ-500 platform. Three independent biological replicates were carried out for the leaves of wild type and EsMYB90 transgenic tobacco, respectively. The methods of getting the clean reads, gene functional annotation, identification of differential expressed genes, and GO and KEGG pathway functional enrichment analysis has been reported and shown in our previous publication [37 (link)]. The DEGs with log₂Fold Change ≥1 (or ≤−1) and Padj ≤ 0.05 were designated as significantly differentially expressed genes.

For each biological replicate of PR, PO and PR, an equal amount of petals at five different opening stages was pooled, respectively. Total RNA was extracted from 1-mg pooled samples of petals from PR, PO and PR obtained by homogenizer (TissueLyzer; Qiagen, Valencia, CA, USA) using the Quick RNA Isolation Kit (Bioteke Corporation, Beijing, China) according to the manufacturer’s protocols. The RNA purity was examined by using the NanoDrop 2000 spectrophotometer (Thermo Scientific, Waltham, MA, USA) and 1% agarose gel electrophoresis. The RNA integrity was checked using an Agilent 2100 Bioanalyzer (Santa Clara, CA, USA) with an RIN (RNA integrity number) > 8.0. The Bioanalyzer results of all RNA samples were listed in Table S10. 6-μg total RNA of three biological replicates from PR, PO and PR were used for library construction. The library construction and the RNA-seq analysis were performed by the Biomarker Biotechnology Corporation (Beijing, China) using an Illumina HiSeq^TM 2500 platform. All sequencing data were deposited in the National Center for Biotechnology Information (NCBI) Sequence Read Archive with accession number SRP109687.

Honey bees larvae treated as described above were used for transcriptomic analysis. Ten larvae from the same plates were pooled together as one replicate. For each experimental group, four or five biological replicates were isolated. The total RNA of each sample was isolated with Quick RNA isolation kit from Bioteke Corporation, China. The RNA integrity and quality were assessed by 1% agarose gel electrophoresis and Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, USA), respectively. The mRNA-Seq libraries were created using NEBNext^® Ultra™ RNA Library Prep Kit for Illumina^® (NEB, Ipswich, Massachusetts, USA) following manufacturer’s recommendations. The RNA-Seq were performed on an Illumina Hiseq 2000 platform (Illumina Inc., San Diego, CA, USA) following the standard Illumina preparation protocol by Biomarker Biotechnology Corporation (Beijing, China).

Total RNA for Illumina sequencing was isolated from shoot tissues of plants grown under salt treatment or control conditions using a Quick RNA Isolation Kit (BioTeke Corporation, Beijing, China). The quantity and quality of the total RNA were assessed using a NanoDrop ND1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA), Qubit 2.0 fluorometer (Life Technologies, Carlsbad, CA, USA) and an Agilent 2100 Bioanalyzer (Santa Clara, CA, USA). The cDNA library was constructed and sequenced by the Biomarker Biotechnology Corporation (Beijing, China). The poly (A) mRNA was enriched via magnetic oligo (dT) beads and then broken into short fragments using an RNA Fragmentation Kit (Beckman Coulter, Brea, CA, USA). These cleaved mRNA fragments were used as templates for first-strand cDNA synthesis using random hexamer primers. Then, second-strand cDNA was synthesized and purified using AMPure XP beads (Beckman Coulter, Brea, CA, USA). These short fragments were ligated to sequencing adapters, and the desired fragments were separated using AMPure XP beads. Next, the purified cDNA fragments were enriched via PCR. Finally, the six cDNA libraries were sequenced using Illumina HiSeq^™ 2500 and 125bp paired-end reads were generated.

Total RNA was isolated from each sample (rhizomes) using a Quick RNA isolation kit (Bioteke Corporation, Beijing, China). Three biological replicates per experimental group were used for sequencing. Agarose gel (1%) electrophoresis was used to assess the contamination and degradation of RNA. The purity and concentration of the isolated RNA were determined using a Nano spectrophotometer (Photometer^®; IMPLEN, CA, USA) and a Qubit^®2.0 Fluorometer Kit (RNA Assay, Life Technologies, CA, USA), respectively, whereas an Agilent 2100 Bioanalyzer system (Nano 6000; Agilent Technologies, CA, USA) was used to evaluate RNA integrity.

Total RNA was extracted from 100 mg petal base of P. rockii at S4 using the Quick RNA Isolation Kit (Bioteke Corporation, Beijing, China). After RNA purity and integrity were checked, full-length cDNAs of PrMYB5 were cloned using the SMARTer RACE 5'/3' Kit (TAKARA Corporation, Beijing, China) according to the manufacturer's protocols. The primers are listed in Supplementary Table 1.
The multiple sequence alignment and phylogenetic tree of PrMYB5 and some anthocyanin biosynthesis and spot formation related R2R3-MYB proteins from tree peony and other plants were performed using the DNAMAN6.0.3.99 software and the MEGA 6.0 software using the neighbor-joining method, respectively.