Rneasy plant mini kit

Leaf samples from the treatments with Al concentration of 0 (L0), 1 (L1) and 4 (L4) mM were collected for RNA-Seq analysis. The Al concentration at 1 mM, was used as the control (CK) because of good root growth at that concentration. Equal amounts of frozen leaves from three individual plants were mixed as one biological replicate. There were three biological replicates for each treatment. Total RNA was extracted using an RNeasy Plant Mini Kit (TaKaRa, Dalian, China), according to the manufacturer’s protocol. The concentration and quality of total RNA were examined using a NanoDrop 2000c spectrophotometer (NanoDrop, Wilmington, DE, USA) and 1% w/v agarose gel electrophoresis. The cDNA libraries were sequenced by a llumina Hiseq Xten platform by the Bioacme Biotechnology Co., Ltd. (Wuhan, China). The high-quality clean reads from each sample were mapped to the reference genome^{48 (link)} using Hisat2.

Using an RNeasy Plant Mini kit (Takara, Dalian, CN), total RNA from all samples was extracted following the manufacturer’s instructions. DNA contamination was eliminated with DNase Ⅰ. RNA concentration was measured using NanoDrop 2000 (Thermo Scientific, Waltham, USA). RNA integrity was assessed using the RNA Nano 6000 Assay Kit of the Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, USA).
A total of 1 μg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEB Next Ultra^™ RNA Library Prep Kit for Illumina (NEB, USA) following the manufacturer’s recommendations and index codes were added to attribute sequences to each sample. Briefly, it involved a series of procedures, including mRNA purification, first and second cDNA strand synthesis, adaptor ligation, PCR amplification, and cluster generation. The library preparations were sequenced on an Illumina Hi-seq Xten platform and paired-end reads were generated. Clean data (clean reads) were obtained by removing reads containing adapter, reads containing ploy-N, and low quality reads from the raw data. At the same time, Q20, Q30, GC-content, and sequence duplication level of the clean data were calculated. All the downstream analyses were based on clean data with high quality.

For study the diurnal expression of SP3D, SP5G, SP5G2, and SP5G3 genes, tomato leaves were harvested every 4 h for 24 h (0, 4, 8, 12, 16, 20, and 24 h), pooled from 3 third leaves of 5-week old plants. Total RNA was extracted using an RNeasy Plant mini kit (Takara, Dalian, China) following the manufacturer’s instructions. cDNA synthesis was performed by using the SuperscriptIII First strand synthesis system (Invitrogen, Shanghai, China) following the manufacturer’s instructions. Real-time PCR was performed using SYBR Premix Ex Taq (Takara, Dalian, China) in a Bio-Rad CFX96 real-time PCR system (Bio-Rad, Hercules, CA, USA). As an internal control gene, actin transcripts were assayed. The primers used are listed in Additional file 1: Table S1. Real-time quantitative PCR was repeated three times, and each time every sample was assayed in triplicate by PCR.