Rnaprep pure plant plus kit polysaccharides polyphenolics rich

Total RNA from azalea leaves was extracted using the RNAprep Pure Plant Plus Kit (Polysaccharides & Polyphenolics-rich) (TIANGEN, Beijing, China). Poly(A) RNA (mRNA) was enriched by oligo(dT) magnetic beads and quantified using the Agilent 2100 Bioanalyzer. Full-length first-strand cDNA was synthesized using a UMI base PCR cDNA Synthesis Kit (BGI, Shenzhen, China). After large-scale amplification, cDNA was used to construct two SMRT cell libraries (0–5 K and 4.5–10 K) using a DNA Template Prep Kit (Pacific Biosciences of California). SMRT sequencing was carried out on the Pacific Bioscience Sequel platform. Subreads were filtered to obtain high-quality consensus transcripts using the SMRT Analysis Server. Blastx (Altschul et al., 1990 (link)) or Diamond (Buchfink et al., 2015 (link)) was used for NR, KOG, KEGG, and Swiss-Prot annotation. The Swiss-Prot database version used for protein search is release 2018_08, and taxonomy used for protein search is Viridiplantae. Release 2018_08 of Swiss-Prot contains 558125 sequence entries, comprising 200328830 amino acids. Blast2GO (Conesa et al., 2005 (link)) with NR annotation results was used for GO annotation.

Total RNA of Korla pear at different developmental stages was extracted from peel tissues using the RNAprep Pure Plant Plus Kit (Polysaccharides & Polyphenolics-rich) (TIANGEN, Beijing, China) with three biological replicates. RNA integrity was assessed using an Agilent 2100 bioanalyzer (Agilent, CA, USA). After the sample passed the quality inspection, two micrograms of total RNA from each sample were used to generate the sequencing libraries using a NEBNext Ultra RNA Library Prep Kit for Illumina. The constructed RNA libraries were sequenced on an Illumina HiSeq-Xten platform in paired-end 150 bp mode in Beijing Novogene Biological Information Technology Co., Ltd. (Beijing, China).

B409 and ZS8 seeds were sown separately in 9 × 9 cm pots containing a mixture of peat, nutrition substance, and vermiculite in a 5:4:1 volume ratio and placed in an AR75L growth chamber (Percival Scientific Inc., Perry, IA, USA) with an 8 h light/16 h dark diurnal cycle. The growth chamber was maintained at a constant temperature of 22 °C and 65% relative humidity. The fourth true leaves of B409 and ZS8 were sampled every 4 h for 1 day at 30 days after sowing (Figure S1D). The first sampling was conducted as soon as the light was turned on. Three biological replicates were obtained per time point, and each biological replicate was composed of four independent plants. The samples were immediately snap-frozen in liquid nitrogen and stored at −80 °C. Total RNA was isolated from 42 samples using an RNAprep Pure Plant Plus Kit (Polysaccharides & Polyphenolics-rich) (Tiangen Biotech Co. Ltd., Beijing, China) according to the manufacturer’s instructions. The RNA quality was detected by 1% agarose gel electrophoresis, and its concentration and purity were determined using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA).

The total RNAs were extracted with RNAprep Pure Plant Plus Kit (Polysaccharides & Polyphenolics-rich) (Tiangen Biotech (Beijing) CO. LTD.). The extracted total RNA was subjected to agarose gel electrophoresis to analyze the RNA degradation and contamination degree. RNA purity was analyzed by Nanodrop. RNA concentration was measured by Qubit. RNA integrity was analyzed by Agilent 2100.
The full-length cDNA library construction was as follows: (1) Use oligo (dT) to enrich mRNA containing polyA; (2) Reverse transcription of mRNA into cDNA using a SMARTer PCR cDNA Synthesis Kit; (3) PCR amplification of enriched cDNA; (4) Use magnetic beads to select fragments for large-scale PCR to obtain sufficient total cDNAs; (5) Full-length cDNA for damage repair, end repair, and connect SMRT dumbbell-shaped adapters to construct a full-length transcriptome library; (6) Digestion with exonuclease to remove the unlinked linker sequences at both ends of the cDNA; (7) Finally, primers and DNA polymerase were bound to form a complete SMRT bell library. RNA isolation and library construction of second-generation transcriptome were performed according to the method described by Qin [45 ].

Total RNA was isolated from sweet potato leaves (leaves obtained from three-month-old plants), stems (stems obtained from three-month-old plants) and roots at five stages (FR, fibrous roots; D1, 1 cm storage roots; D3, 3 cm storage roots; D5, 5 cm storage roots; and D10, 10 cm storage roots) by a RNAprep Pure Plant Plus Kit (Polysaccharides & Polyphenolics-rich; TIANGEN, Beijing, China). Reverse transcription and qRT-PCR were performed as described by Tang et al. [20 (link)]. An adaptor (5′ GTC GTA TCC AGT GCA GGG TCC GAG GTA TTC GCA CTG GAT ACG AC 3′) was added to the 3′ end of the miRNAs through a reverse transcription program; the reverse transcription of the miRNAs was performed by a PrimeScript™ RT reagent kit with gDNA Eraser (TaKaRa, Dalian, China). qRT-PCR was performed by using TB Green™ Premix Ex Taq™ II (TaKaRa, Dalian, China) and CFX96™ Real-Time System (Bio-Rad, Hercules, CA, USA) with the following procedures: 95 °C for 30 s, then 95 °C for 5 s and 60 °C for 40 s for 40 cycles. The primers used for miRNA qRT-PCR analysis are listed in Table S2. Sweet potato ARF (ADP-ribosylation factor) was used as the reference gene for the normalization of gene expression [22 (link)].

Real-time quantitative RT-PCR was performed, as previously described by Li et al.^{35 (link)}. For tissue-specific expression analysis, total RNA was isolated from buds, stems, vascular bundles, first leaves, leaf veins of the first and third leaves, leaf veins of the third and fifth leaves, leaf veins of the fifth leaf, and roots using an RNAprep Pure Plant Plus Kit (polysaccharides & polyphenolics-rich) (TIANGEN, Beijing, China). The RNA concentration and integrity were evaluated via a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA) and confirmed via gel electrophoresis. Total RNA (1 μg) was reverse-transcribed with Oligo dT primer using a HiScript^® II One Step RT-PCR Kit (Vazyme, China). qRT-PCR was performed on a Bio-Rad CFX96 in conjunction with SYBR Green I dye (Vazyme, China), while qPCR was performed on a Bio-Rad CFX96 Real-Time PCR Detection System (Bio-Rad, Hercules, CA). Primers were designed using Primer-BLAST (http://www.ncbi.nlm.nih.gov/tools/primer-blast/). The results were normalized to those of a housekeeping gene, CsGAPDH, and calculated by the comparative Ct method^{36 (link)}. The primers used for real-time quantitative RT-PCR are listed in Supplementary Table S1.