Dnase free rnase

Total RNA was extracted using GenElute mammalian total RNA miniprep kit (Sigma) according to the manufacturer’s protocol. RNA samples were treated with RNase-free DNase (Sigma). RNA integrity was assessed by NanoDrop, Qubit assay, and/or using an Agilent 2100 bioanalyzer per each manufacturer’s specifications. Sample amounts were normalized, and 1,000 ng was used for library preparation using the NEB Ultra RNA Library Preparation kit per the manufacturer’s instructions. Library quality control (QC) and quantitation were performed on all individual libraries by the Qubit assay and by using the Agilent 2100 bioanalyzer. Libraries were normalized and pooled via Qubit measurement. The final pool was quantitated via quantitative PCR (qPCR). Sequencing was performed on the Illumina HiSeq2500 rapid-run mode on one flow cell (two lanes) per the system manufacturer. Raw RNA-seq data were processed, normalized, and mapped to the human reference genome (hg19) using CLC Genomics Workbench 8 (Qiagen). Differentially expressed genes were identified using DESeq2 (38 (link)) with the indicated significance cutoffs. Hierarchical clustering was performed using Cluster 3.0/Java Treeview and heat maps were generated using MeViewer software (17 (link)).
Pathway analysis was performed using MetaCore by GeneGo, with a statistical cutoff of P < 0.001 applied for pathway enrichment.

Total RNA was isolated using an RNA Purification Kit (Thermo Fisher Scientific). Total RNA (200 ng) was treated with RNase-free DNase (Sigma Aldrich) for 15 min. Following the inactivation of DNase with Ethylenediaminetetraacetic acid (EDTA) and heating, RNA was reverse transcribed using a First Strand cDNA Synthesis Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. Quantitative real time polymerase chain reaction (RT-PCR) (qPCR) was performed with cDNA samples using the Power SYBR Green Master Mix and Mic qPCR Cycler (Bio Molecular Systems). The relative mRNA level was calculated as values of 2^{(Ct(β-actin) − Ct(gene of interest))}. For data presentation, the mRNA level in control cells was set to 1. The sequences of the forward and reverse primers are as follows: β actin, 5’-GGCTGTATTCCCCTCCATCG-3’ and 5’-CCAGTTGGTAACAATGCCATGT-3’; Ccna1, 5’-TGATGCTTGTCAAATGCTCAGC-3’ and 5’-AGGTCCTCCTGTACTGCTCAT-3’; Ccna2, 5’-GCCTTCACCATTCATGTGGAT-3’ and 5’-TTGCTGCGGGTAAAGAGACAG-3’.

A total of 150 mg of root sample of IR64 and LS was homogenized in liquid nitrogen using pestle and mortar. Total RNA was isolated using Trizol reagent (Invitrogen, http://www.invitrogen.com, last accessed on 20 July 2021), as per the manufacturer’s instructions. RNase-free DNase (Sigma-Aldrich, USA) was used to remove the genomic DNA and 2 µg of RNA was used to synthesize cDNA in a total volume of 10 µL reaction using the iScript cDNA synthesis kit (Bio-Rad, Hercules, CA, USA) as per the manufacturer’s recommendations.

Total RNA was extracted using GenElute mammalian total RNA miniprep kit (Sigma) according to the manufacturer’s protocol, treated with RNase-free DNase (Sigma), and reverse transcribed using iScript cDNA synthesis kit (Bio-Rad). For each sample, 1 µg total RNA was used for cDNA synthesis. RT-qPCR was performed using iQ SYBR green supermix (Bio-Rad) in an Applied Biosystems StepOne real-time PCR machine. Gene expression was calculated using a modified ΔC_T method (C_T stands for threshold cycle) based upon normalization to human actin. Primer sequences can be found in Table S1 in the supplemental material.

The sampled plant leaves were grounded to fine powder with mortar and pestle in liquid nitrogen, and 100 mg of the material was used for RNA isolation. Total RNA was extracted using Trizol reagent (Invitrogen, http://www.invitrogen.com), as per the manufacturer’s guidelines. Isolated RNA was treated with RNase free DNase (Sigma-Aldrich, USA) to remove genomic DNA contamination. Purity and concentration of RNA samples was measured using NanoDrop (Thermo Scientific) and integrity was checked on agarose gel electrophoresis. RNA samples with 260/280 ratio between 1.9 and 2.1 were used for subsequent experimentation. First strand cDNA synthesized using iScript cDNA synthesis kit (BioRad) according to manufacturer’s instructions in a total volume of 20 μl containing 2 μg total RNA. RNA extraction and cDNA synthesis from all samples were performed for two biological replicates. The cDNA solution was 10 times diluted with nuclease-free water and aliquots were stored at -20°C till qRT-PCR.

Diethyl phthalate 99.5% (DEP), benzyl butyl phthalate 98% (BBP), and bis-(2-ethylhexyl) phthalate 98% (DEHP) were acquired from Sigma (Spain). Stock solutions for each compound were prepared at 10.78 mg/mL for BBP, 11.44 mg/mL for DEP, and 965.3 mg/ml for DEHP. The stock solutions were prepared in acetone for BBP and DEP, while DEHP was diluted in ethanol. Exposure solutions were prepared by 1:10,000 dilution of these stocks in artificial pond water (see the Treatment section below).
TRIzol and M-MLV enzyme were obtained from Invitrogen (Germany), oligonucleotide dT18 primer and gene-specific primers were supplied by Macrogen (Korea), RNase-free DNase was purchased from Sigma, DNA polymerase and dNTPs were obtained from Biotools (Spain), and EvaGreen was purchased from Biotium (USA).