Fruit mate for rna purification

RNA was extracted using a Maxwell^® 16 Automated Purification System (Promega, Madison, WI, USA) with Fruit-mate™ for RNA Purification (Takara Bio, Kusatsu, Japan). The RNA concentration was determined with a Nanodrop 1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), and stored at −80 °C.
For reverse transcription we used 100 ng total RNA with the ReverTra Ace^® qPCR RT Kit (Toyobo, Osaka, Japan) in accordance with the manufacturer’s protocol. The cDNA solution was diluted two-fold and stored at −20 °C.
Real-time PCR was carried out in a LightCycler Nano or 480 (Roche Diagnostics, Basel, Switzerland) with the THUNDERBIRD^® SYBR^® qPCR Mix (Toyobo). The PCR assay was prepared to 20 µL total volume containing 10 µL THUNDERBIRD^® SYBR^® qPCR Mix, 2.0 µL cDNA solution, and 10 µM gene-specific primers. The gene-specific primer sequences are listed in Table S1. The thermal cycling procedure was as follows: 95 °C for 30 s, three-step amplification consisting of 40 cycles of 95 °C for 15 s, 60 °C for 30 s, and 72 °C for 30 s, a pre-melt hold at 95°C for 10 s, and melting at 60 °C to 97 °C at a rate of 0.1 °C/s. All target gene data were normalized against EF1α transcript levels.

The leaves and internodes of each plant species were homogenized in an SK mill (SK-200, Tokken, Kashiwa, Japan) after freezing with liquid nitrogen. Total RNA extraction from the pulverized samples was performed with a NucleoSpin RNA Plant Kit (TaKaRa, Otsu, Japan) after pretreatment with a Fruit-mate for RNA Purification (TaKaRa) according to the manufacturer’s instructions.

Two local varieties of Tibetan hulless barley, XQ754 and Nimubai (used and known as tribute barley), were conserved by the Tibet Academy of Agricultural and Animal Husbandry Sciences. Nimubai has a higher amylose content (33.9%) and β-glucan content (7.5%) as compared to XQ754, which had 27.2% amylose and 6.0% β-glucan (data collected from 2009–2010 in Chengdu). The hulless barley plants were cultivated in October, 2010 and grown under normal conditions in the three fields in Chengdu, Sichuan Province of China.
Grains of Nimubai and XQ754 plants were sampled at 5, 10, 15, 20, and 25 days after pollination (dap) for RNA extraction. Each sample consisted of grains from nine individuals. Total RNA was extracted from the grains using Trizol Reagent (Takara) and Fruit-mate for RNA purification (Takara), according to the manufacturer's instructions. The concentration and quality of RNA samples were determined using a Nano Drop 2000 micro-volume spectrophotometer (Thermo Scientific, Waltham, MA, USA). Equal amounts of RNA from each sample of the identical accessions were pooled to construct two cDNA libraries [26] (link), [27] (link).

Fifteen fresh berries of Koshu and Pinot Noir were randomly collected from each grapevine at two weeks post véraison and at harvest. Skins were peeled off from the berries by using tweezers. The skins were placed in a mortar containing liquid nitrogen and homogenized with a pestle. Total RNA isolation from the pulverized skins was performed with a Fruit-mate for RNA Purification (Takara, Otsu, Japan), followed by a NucleoSpin RNA Plant (Takara) for RNA purification according to the manufacturer’s instructions.

Five leaf samples from a single plant were rapidly frozen in the presence of liquid nitrogen and kept at −70 °C for further experiments. Frozen leaf tissues were ground in liquid nitrogen with a mortar and pestle. We extracted total RNA using two kits: Fruit-mate for RNA Purification (Takara, Shiga, Japan) and the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany) according to manufacturer’s instructions. The quality and quantity of extracted total RNA were checked. Only high quality total RNA was used for the library preparation for RNA-Seq.

Total RNA samples were extracted from seeds of IM, HN, HJJ, JJJ, and HoJJ harvested 20 DAP using Fruit-mate for RNA Purification (Takara, Otsu, Japan) and Plant RNA Purification Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturers′ instructions. The first-strand cDNAs were synthesized with amfiRivert cDNA Synthesis Platinum Master Mix (GenDEPOT, Barker, TX, USA). To amplify the coding regions of CYP75B3 and CYP75B4 without the stop codon, gene-specific primers were designed based on the two sequences in the public database. PCR was performed in 50-μL reactions containing 1 µL of four-fold-diluted cDNA, 0.4 µM of each primer (Table 2), PrimeSTAR HS DNA polymerase, and 5× reaction buffer (Takara) under the following conditions: 98 °C for 2 min; 30 cycles at 98 °C for 10 s, 60 °C for 15 s, 72 °C for 2 min; and a final extension at 72 °C for 5 min. The amplicons were subcloned into the pENTR-SD/D-TOPO vector (Invitrogen) via directional cloning technology and verified by DNA sequencing.

Three biological replicates were used for library constructions and sequencing. Each sample was initially treated with Fruit-Mate for RNA purification (Takara) and thereafter the total RNA was isolated with TRIzol Reagent (Thermo Fisher Scientific). An Illumina TruSeq RNA Sample Prep Kit (Illumina) was used to build cDNA libraries. The generated cDNA libraries were sent to a HiSeq2500-PE125 platform (Illumina) to obtain sequence reads. Based on the genome sequences of maize (B73, RefGen_v4), the Tophat and Cufflinks packages were used to assemble the transcripts and to identify differentially expressed genes (DEGs) using DEGseq2 (Trapnell et al., 2012 (link); Love et al., 2014 (link)), and only genes whose levels were at least 2-fold changed with statistical significance (false discovery rate <0.05) were considered to be DEGs. Gene Ontology (GO) analysis and graphing were performed on Omicshare (https://www.omicshare.com/tools/), ImageGP (http://www.ehbio.com/), and agriGO (http://systemsbiology.cau.edu.cn/agriGOv2).