Transzol plant kit

The samples of maize B73 tissues or organs at specific developmental stages were collected according to the methods as described previously [49 (link)]. Total RNA was isolated from the harvested samples using the TransZol Plant kit (TransGen, Beijing, China), then subjected to the cDNA synthesis using the TransScript All-in-One First-Strand cDNA synthesis kit (TaKaRa, Beijing, China) according to the manufacturer’s instructions. Diluted cDNA samples were used for real-time PCR amplification in 10 μL reaction volumes with specific primers and SYBR Green PCR Master Mix (TaKaRa, Beijing, China). Quantitative real-time PCR was run on the Applied Biosystems 7500 system (Prism^® 7500, Carlsbad, America) using the following reaction conditions: 95 °C for 1 min followed by 45 cycles of 95 °C for 10 sec and 60 °C for 15 sec. ZmACTIN gene was used as the internal control, and the relative expression level of each gene were calculated using the 2^−∆∆CT method [50 (link)]. Primers used in this study were listed in Table S2.

Total RNA was extracted from various plant tissues of Nipponbare, as well as from developing grains (0, 7, 14, 21, 28, 35 and 42 days after flowering; DAF) and germinating seeds (4‐, 8‐, 12‐, 18‐, 24‐, 36‐ and 48‐h imbibition), using the TransZol Plant kit (Transgen, www. transgen.com), according to the protocol by the manufacturer. The first‐strand cDNA was synthesized with random oligonucleotides using the HiScript^® II Reverse Transcriptase system (Vazyme Biotech Co., Ltd). qRT‐PCR was carried out in a total volume of 20 μL containing 2 μL of cDNA, 0.4 μL gene‐specific primers (10 μm), 10 μL SYBR Green Mix and 7.2 μL of RNase free ddH₂O, using the Roche LightCycler480 Real‐time System (Roche, Swiss Confederation). The PCR conditions were as follows: 95 ^°C for 5 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 30 s. The rice OsActin and 18S rRNA genes were used as internal controls. Primers used for qRT‐PCR are listed in Table S1. Normalized transcript levels were calculated using the comparative CT method (Livak and Schmittgen, 2001). Three biological replications were performed.

Total RNA was isolated and 2 μg of RNA in each sample was treated with DNase I to destroy traces of possible DNA contamination using the TransZol Plant kit (TransGenBiotech, Beijing, China) following the manufacturer’s protocol. Quality of purified RNA was checked by agarose gel electrophoresis and integrity of RNA was checked by RNA 6000 Nano kit (Agilent, Santa Clara, CA, USA). RNA concentration was measured by NanoDrop 2000 spectrophotometer (Thermo Scientific, Waltham, MA, USA). The RNA-seq cDNA libraries were constructed using TruseqTM RNA sample prep kit (Illumina, San Diego, CA, USA). Sequencing was running on an Illumina HiSeq4000 sequencer, and data analyses were performed on Majorbio Cloud platform (Majorbio, Shanghai, China).

Genomic DNA was extracted from 3 g of seedlings grown in the dark at 25 °C for 7 days by the CTAB method [48 ]. Total RNA was extracted from five whole grains at different developmental stages with the TransZol Plant Kit (TransGenBiotech).

Total RNA was extracted from plant materials using a TransZol Plant kit (ET121-01, Transgen Biotech, Beijing, China) according to the manufacturer’s instruction. Digestion of gDNA in each sample was conducted using DNase I (2212, TaKaRa, Dalian, China). The integrity and purity of RNA was confirmed using 1.2% non-denaturing agarose gel electrophoresis and the Agilent 2100 RNA 6000 Kit (Agilent Technologies, Santa Clara, CA, USA).

Total RNA was extracted from 0.2 g of tested material using a TransZol Plant kit (TransGen Biotech, Co., Ltd., Beijing, China). The specific method was implemented according to the kit instructions, and the RNA precipitation was dissolved in sterile water treated with 30 μL of DEPC. The total RNA was submitted as a template to synthesize cDNA using TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix kit (TransGen Biotech Co., Ltd.) according to the instructions.

Total RNA was extracted from approximately 80~100 mg powder of WT and osipms1 seeds after 8‐h imbibition using the TransZol Plant kit (Transgen, www.transgen.com) according to the manufacturer's protocol. Construction of cDNA libraries and HiSeq2500 sequencing were performed at Novogene Biotechnology Co., Ltd., Beijing, China. FastQC was performed to estimate the quality of raw reads ( http://www.plob.org/2013/07/16/5987.html). The adapter sequences were trimmed, and the low‐quality reads with Q ≤ 20 from the 5′ and 3′ ends of the remaining reads were filtered. Then, the clean reads with 21–49 bp length were mapped onto the Nipponbare reference genome (MSU Rice Genome Annotation Project Release 7) using Tophat version 2.0.12 (Kim et al., 2012). Levels of gene expression were quantified in terms of FPKM (fragments per kilo base of exon per million) using RSEM version 1.1.11 (Li and Dewey, 2011). The log2‐fold changes of gene FPKM were calculated in 8‐h imbibed seeds and comparisons were made between osipms1a and WT. The differentially expressed genes (DEGs) with a padj (P‐adjusted) <0.05 were selected for further pathway analysis. Gene Ontology (GO) and KEGG pathway analyses were performed through GOseq (Young et al., 2010) and KOBAS (2.0) with a significant level of false discovery rate (FDR < 0.05; Xie et al., 2011). Three biological replications were performed.