Transzol kit

Total RNA extraction of finger millet leaf tissue was conducted using a TransZol kit (TransGen Biotech, Inc., Beijing, China). Sequencing libraries were constructed using an NEBNext^® Ultra^™RNA Library Prep Kit for Illumina^® (NEB, Ipswich, MA, USA). There were three biological replicates in the N1, N2 and N3 group. Paired-end non-strand specific libraries (200-250bp) were constructed and sequenced based on the Illumina HiSeq^™ X-Ten platform (BGI, Shenzhen, China). FastQC was used to control the quality of sequencing data [17 (link)] and Trimmomatic-0.36 was used to remove low-quality sequences [18 (link)]. Bowtie2 aligns clean reads to the genome sequence [19 (link)], followed by the use of RSEM to calculate the expression level of each sample [20 (link)]. The resulting quantifications were expressed as fragments per kilobase million (FPKM) [21 (link)]. The differentially expressed genes (DEGs) were screened using DEseq2 based on an absolute fold change value of |log₂foldchange| > 1 and an adjusted P value ≤ 0.01 [22 (link)].

Total RNA in mouse liver from each group was extracted using TransZol kit (Beijing TransGen Biotech Co., Ltd., Beijing, China), according to the manufacturer’s protocol and RNA concentration was quantified with a Nanodrop spectrophotometer. The first strand cDNA was synthesized from 1 µg of RNA by using First-Strand cDNA Synthesis Kit (Beijing Cowin Biotech Co., Ltd., Beijing, China). Quantitative real-time polymerase chain reaction (q-PCR) was used to determine the expressions of genes, such as CYP1A1, CYP2E1, CYP3A11, and β-actin by using UltraSYBR Mixture (Beijing Cowin Biotech Co., Ltd., Beijing, China). The primers of q-PCR are described in Table 1. The expression of individual genes was performed using the ABI 7500 Real-time PCR system. The amplifications for all reactions were performed as following: 10 min 95 °C, followed by 40 cycles of 30 s at 95 °C and 40 s at 60 °C. Results were calculated using the relative 2^−ΔΔCt method [53 (link)].

Total RNA was isolated from leaves using TransZol kit (TransGen, China) and then was treated with DNase I (Takara, China). Purified RNA was reverse transcribed in a 10 µl reaction using an oligo-dT18 primer and first-strand cDNA was synthesized using PrimerScript RT reagent Kit according to the manufacturer’s instructions (Takara, China). Q-PCR amplifications were performed on a 96-well plate with a Bio-Rad CFX96 real-time PCR system. Each reaction contained 10 µL of SYBR Premix Ex Taq (Takara, China), 2 µL of cDNA samples (diluted to 5ng µL⁻¹) and 0.8 µL of gene-specific primers (10 µM) in a final volume of 20 µL. Gene-specific primers were designed according the conserve region of each gene family or subfamily (Table S1). The thermal profile consisted of one cycle at 95 ° C for 30 s followed by 40 cycles of 95 °C for 5 s, 57 °C for 30 s and 72 °C for 20 s. The reference gene actin7 was used to normalize for differences of the total RNA amount. The relative quantities of gene expression for sample comparison were calculated using the comparative Ct (2^−ΔΔCt) method (Wong & Medrano, 2005 (link)). In total, there were two technical replicates for each biological replicate with three biological replicates in each treatment.

The expression level of candidate genes in two Pb-tolerant (HANNA and III-229) and two Pb-sensitive (6024–1 and EH3143) genotypes were evaluated by quantitative real time PCR (qRT-PCR). Total RNA was extracted from seven-day-old radicles grown under normal (0 mg/L) or Pb stress (100 mg/L) condition using TransZol kit (Trans Gene Biotech). A total amount of 500 ng RNA was used to synthesize first strand cDNA using HiScript® II Q Select RT SuperMix for qPCR (+gDNA wiper) kit (Vazyme Biotech). The gene copy specific primers of candidate genes were designed using Primer Premier 5 (Additional file 9: Table S5). The qRT-PCR assay was carried out using LightCycler® 480 SYBR Green I Master kit (Roche Life Science) in LightCycler® 480 qPCR machine (Roche Life Science) according to the manufacturer instructions. Data were collected from three technical replicates. The relative expression level was normalized by BnACTIN7 using a △CT method (Li et al., 2017). The tukey test was employed for differentiation analysis on relative gene expression level between accessions and treatments.

Total RNA was extracted from the EBZ and NEZm of P. pratensis leaf using TransZol kit (TransGen, China). DNA contamination was removed with RNase-free DNase I (Takara, China). RNA quality and quantity were assessed by absorption at 260 nm/280 nm, and gel electrophoresis. Briefly 2.5 μg of total RNA was enriched for Poly-A using NEBNext Poly (A) mRNA Magnetic Isolation Module (NEB, E7490). Transcriptome library for sequencing was constructed according to NEBNext mRNA Library Prep Master Mix Set for Illumina (NEB, E6110) and NEBNext Multiplex Oligos for Illumina (NEB, E7500). The prepared library was quantified using Library Quantification Kit-Illumina GA Universal (Kapa, KK4824) and validated for quality by running 1.8 % agarose gel electrophoresis. The library products were sequenced via Illumina HiSeq™2500 sequencer.

Twelve up-regulated unigenes in the NEZm with potential roles in cuticular wax deposition were chosen for validation using qRT-PCR. Total RNA was extracted from the EBZ and the NEZm of P. pratensis leaf using TransZol kit (TransGen, China). DNase-treated RNA was used to synthesize first strand cDNA by using SuperScript II reverse transcriptase (Invitrogen, China). The gene names and primers used for qRT-PCR are liste in Additional file 2: Table S6. The quantitative reaction was performed on the CFX96 Real-Time PCR Detection System (Bio-Rad) using the SYBR Premix Ex TaqII(Takara, China). qRT-PCRs were performed as follows: 95 °C for 30 s, 40 cycles of 95 °C 5 s, 59 °C 30 s, and 72 °C 15 s. CFX Manager software (Bio-Rad) was used for data analysis. Expression levels of the selected unigenes were normalized to that of Elongation factor 1 (eEF1-a), an internal reference gene [52 (link)]. The relative expression levels of target genes were calculated with the 2^-ΔΔCt method [53 (link)]. All the experiments were repeated using three biological and three technical replicates and the data were analyzed statistically.

Total RNA extraction was carried out using the TransZol kit (TransGen Biotech, Inc., Beijing, China). cDNA reverse transcription refers to use of the HiScript^®IIQ RT SuperMix for qPCR (+gDNA wiper) vazyme kit (Vazyme, Piscataway, NJ, United States). quantitative polymerase chain reaction (qRT-PCR) was carried out in LightCycle ^∗ 96 Real-Time PCR System (Roche, Basel, Switzerland) with 25 μL reaction system. Three biological repeats and three technical repeats were used to calculate the relative quantification according to the Ct values collected by the instrument. The formula is: 2^−△△Ct =  2^{−[(Target gene control Ct−Target gene sample Ct)−(Reference gene control Ct−Reference gene sample Ct)]}. The actin gene Capana04g001698 was used as reference gene which was selected from pepper. The primers of six CaR2R3-MYB DEGs and four CBGs were developed by GenScript Real-time PCR (TaqMan) Primer and Probes Design Tool⁶, which were listed in Supplementary Table 3.