Rnaclean kit

The total RNA was extracted from cell samples with TRIzol reagent (Invitrogen, CA, USA). The RNA was purified with RNA clean kit (TIANGEN, PD412, Beijing, China) according to the manufacturer's instructions, and RNA content and purity of the cell samples were measured with NanoDrop ND-1000 (NanoDrop, Wilmington, DE, USA). We evaluated the integrity of RNA using Bioanalyzer 2100 (Agilent, CA, USA), where RIN > 7.0 is consistent with downstream experiments. The RNA integrity was also confirmed by electrophoresis with denaturing agarose gel. In two rounds, Dynabeads Oligo (dT)25–61,005 (Thermo Fisher, CA, USA) was used to purify poly(A) RNA from 1 μg total RNA. Using the Magnesium RNA Fragmentation Module (NEB, cat.e6150, USA), poly(A) RNA was fragmented and reverse-transcribed using the SuperScriptTMII Reverse Transcriptase (Invitrogen, cat.1896649, USA) to generate cDNA. Next, a paired-end cDNA library was created with an average insert size of 300 ± 50 bp. In accordance with the standard protocol, we used the Illumina Novaseq™ 6000 (LC Bio Technology CO., Ltd. Hangzhou, China) to perform double-end sequencing.

Nostoc at the mid-exponential growth phase was cultured with or without arsenic for 6 h. Total RNA was extracted from Nostoc cells treated with As(III) (0, 1, 5, 10, 40, and 100 μM) or MAs(III) (0, 0.2, 1, 3, 6, and 12 μM) by using TRIzol reagent (Invitrogen Life Technologies, Gaithersburg, MD, USA) following the manufacturer’s recommendations. Contaminating genomic DNA was removed from total RNA using DNase I (Promega, Madison, WI, USA) at 37°C for 45 min. The total RNA was further purified with RNA clean kit (Tiangen, Beijing, China). About 40 ng purified RNA was used for RT-qPCR with GoTaq^® 1-Step RT-qPCR System (Promega, Madison, WI, USA) in a 20-μL volume. The primers for NsarsM and NsarsI were listed in Table 1. qPCR was performed on a LightCycler 480 (Roche Applied Science, Indianapolis, IN, USA). Each reaction was carried out in triplicate with the housekeeping gene rnpB as the internal standard (Vioque, 1992 (link); Latifi et al., 2005 (link)). The PCR efficiencies of the targets (NsarsM and NsarsI) and reference (rnpB) were calculated from the slope of their standard curves (E = 10^[-1/slope]), respectively, and the relative transcript levels of NsarsM and NsarsI were calculated using the formula (E_target)^ΔCptarget^{(control-sample)} × (E_ref)^ΔCpref^{(sample-control)} (Pfaffl, 2001 (link)).

To confirm the RNA-Seq results, we selected several upregulated and downregulated genes for qRT-PCR validation. After RNA extraction, genomic DNA was removed using DNase I (TaKaRa, Japan) and purified with an RNA clean Kit (TIANGEN, China). Reverse transcription was performed using an iScriptTM cDNA Synthesis Kit (Bio-Rad, America), and SsoFast™ EvaGreen® Supermix reagents (Bio-Rad, America) were used in a Bio-Rad CFX96 real-time fluorescence quantitative PCR instrument. qRT-PCR was performed with the following protocol: 95°C for 3 min, followed by 40 cycles of 95°C for 30 s, 59°C for 30 s, and 72°C for 10 s. Actin was used as a reference gene, and relative quantification was performed using the 2^−ΔΔCt method. The primers for actin were as follows: forward, 5'-GTAGGAAATGGCTGACGGTG-3' and reverse, 5'-ATGCTAGGGAAAACAGCCCT-3'. The primers used to verify benzoxazinoid synthesis gene expression are listed in Supplementary Table 1.

Total RNA was extracted from the hair roots of six pairs of full-sib individuals using a buffer containing ß-mercaptoethanol and guanidine that was provided in an RNAiso Plus kit in accordance with the manufacturer’s instructions (Takara Biotechnology Dalian, Co. Ltd., China). RNA was eluted with 40 uL of RNase-free water, and the quantity and purity of total RNA were measured by using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, U.S.). Samples with purity (A260/A280) of 1.8–2.0 were used in the subsequent experiment. Total RNA was purified using an RNAclean Kit (TIANGEN BIOTECH Co., Ltd, Beijing, China). The samples for small RNA sequencing were prepared in accordance with the instructions that were provided by the manufacturer of the Illumina sequencing kit.

S. cerevisiae CEN.PK113-5D and ADR1_ 1802 were incubated at 30°C at 200 rpm. When the OD₆₀₀ reached approximately 1.0, 4 g/L of furfural was added to the medium, and the time was defined as 0 h. After incubation for 2 h, the cells were collected by centrifuging at 4,000 r/min at room temperature for 2 min. Cells obtained from cultures free of furfural at 0 and 2 h were taken as controls. The primers used for qRT-PCR analysis were listed in Table 2. Total RNA was isolated according to previously described protocol (Liu and Slininger, 2007 (link)), and then purified using the RNA clean kit (Tiangen Biotechnology Co., Ltd., Beijing, China). The reverse transcription reaction was carried out using the procedure described by Lewis et al. (2009) (link). HiScript II QRT SuperMix (Norwich Biotechnology Co., Ltd.) and ChamQ universal SYBR qPCR Master Mix (Norwich Biotechnology Co., Ltd.) were applied for qRT-PCR. The qRT-PCR data were analyzed using the method described by Liu et al. (2009) (link). The reference gene used for quantitative PCR is ENO1.

Total RNA was isolated from the leaves of the three replicate plants in each treatment using the Qiagen RNAeasy kit (Qiagen China, Shanghai, China) and purified using an RNAclean Kit (Tiangen Biotech (Beijing) CO. LTD., China) following the manufacturer’s instructions. The integrity of the RNA was monitored on 1% agarose gels, and the purity was checked using a NanoPhotometer^® spectrophotometer (Implen, Westlake Village, CA, USA). The RNA concentration was determined using a Qubit^® RNA assay kit with the Qubit^® 2.0 fluorometer (Life Technologies, San Francisco, CA, USA). A total amount of 3 µg high-quality RNA per sample was used for subsequent RNA sequencing. The cDNA library was constructed for each of the nine RNA samples and sequenced on the Illumina HiSeq 2500 platform (Illumina Inc., San Diego, CA, USA). Before assembly, the adapter sequences, poly N, and low-quality reads were removed from the raw data. An index of the reference genome (directly downloaded from https://phytozome.jgi.doe.gov/pz/portal.html#!bulk?org=org_egrandis, accessed on 19 August 2020) was built using Bowtie v2.0.6 and the paired-end clean reads were aligned to the reference genome using TopHat v2.0.9 [11 (link)]. Then, the mapped reads of each sample were assembled using both Scripture (beta2) [12 ] and Cufflinks (v2.1.1) [13 (link)].

Total RNA was extracted from control and exogenous-inducer-treated samples using the RNA prep Pure Plant Kit (Tiangen, China) based on the manufacturer’s manual. DNA was then removed using the RNA clean Kit (Tiangen, China). The quality and quantity of total RNA was assessed with the Agilent 2100 Bioanalyzer (Agilent Technologies, CA, USA). The library construction and RNA-Seq assay were performed by the Biomarker Biotechnology Corporation (Beijing, China). Poly-(A)-containing mRNA was purified from the total RNA using oligo (dT) magnetic beads and Oligotex mRNA kits (Qiagen, Hilden, Germany), following the manufacturer’s instructions. Fragmentation was carried out using divalent cations. Fragmentary RNAs were used as the template for first strand cDNA synthesis by a cDNA preparation kit. The cDNA was synthesized using RNase H and DNA polymerase I. Then, cDNAs were subjected to end repair, phosphorylation and ligation to sequencing adapters. Afterward, the products enriched by PCR amplification were purified through 2% agarose gelelectrophoresis and quantified by TBS380 (Picogreen). Finally, cDNA libraries were subsequently sequenced using an Illumina HiSeq 4000 platform.