Dnase 1 turbo dna free kit

Three independent cultures for strains HTABWT, YMC42, YMC43, YMC64 and YMC70 were grown to mid-log phase. A total of 3 × 10⁸ cells per culture were collected and washed with nuclease-free water (Sigma). Total RNA was isolated using RiboPure-Yeast Kit (Life Technologies). To eliminate residual DNA, total RNA was treated with 2 μl DNase I (TURBO DNA-free Kit; Life Technologies) for 45 min at 37 °C. RNA was digested once more using DNase I (RNase-Free DNase set; Qiagen) for 10 min at room temperature before purification using the RNeasy Kit (Qiagen). Before the library construction for RNA-seq, the quality of RNA was ascertained using Bioanalyzer (Agilent), and the RNA integrity number (RIN) for the samples ranged from 6.8 to 8.1. The Ribo-Zero Magnetic Gold Kit (Yeast) was used to remove ribosomal RNA from total RNA used for stranded RNA-seq. Library construction was performed using the Illumina TruSeq RNA Sample Preparation Kit version 2 and sequenced using the Illumina Hiseq2000 sequencer.

L. monocytogenes was grown in BHI at 37°C under constant agitation until reaching an optical density at 600 nm (OD₆₀₀) of 0.9, pelleted, and frozen at −80°C. RNAs were extracted as previously described (72 (link)). For each sample, 10 µg of RNA was treated with DNase I (Turbo DNA-free kit, Ambion). RNAs (500 ng) were reverse transcribed with Quantiscript reverse transcriptase (QuantiTect reverse transcription [RT] kit; Qiagen). Quantitative RT-PCRs (qRT-PCRs) were carried out using oligonucleotides qPCR-orfX-for, qPCR-orfX-rev, qPCR-gyrA-for, qPCR-gyrA-rev, qPCR-rpoB-for, and qPCR-rpoB-rev (Table 1), and the products quantified with SYBR green master mix on a C1000 Touch CFX384 machine (Bio-Rad). The expression levels of orfX were normalized to those of L. monocytogenes rpoB and gyrA genes, and the fold changes in expression were calculated using the cycle threshold (ΔΔCT) method. All samples were evaluated in triplicate and in at least three independent experiments.

For each cell line, a nucleus-enriched RNA fraction was isolated from 20 million cells, as detailed in Fort and colleagues^{46 (link)}. Briefly, cells were first lysed in chilled lysis buffer (0.8 M sucrose, 150 mM KCl, 5 mM MgCl₂, 6 mM 2-mercaptoethanol, and 0.5% NP-40) and centrifuged for 5 min at 10,000×g (4 °C). Nuclei pellets were washed twice with lysis buffer before resuspension in TRIzol Reagent (Life Technologies). The RNeasy kit (Qiagen) was used according to the manufacturer’s protocol to extract nucleus-enriched RNA fractions. During the RNA purification process, samples were treated with DNase I (TURBO DNA-free kit, Ambion) following the manufacturer’s recommendations.

Total RNA from seedling root and shoot, flag leaf and panicle was isolated using TRIzol reagent (Invitrogen). The quantitative and qualitative analysis carried out using spectrophotometer (Nano Drop Nd-1000 UV/Vis Spectrophotometer) and 1.2% agarose gel, respectively. Of the total RNA treated with DNase I (TURBO DNA-free ™ Kit (Ambion), 4.5 μg was reverse transcribed using Superscript III Reverse Transcription kit as per manufacturer’s instructions (Invtrogen). The sequence of primers used for qRT-PCR analysis as provided in Table S4. Quantitative PCR (qPCR) was performed with three biological and three technical replicates with appropriate primers using PoweUpSYBR^®green Master Mix as per manufacturer’s instructions (Thermo Fisher, USA). qRT-PCR was carried out in StepOne real time PCR machine (Applied Biosystems). Expression data were normalized using endogenous control gene OsUBIQUITIN5 (UBQ5). Relative fold change was calculated using the 2^−ΔΔCt method [58 (link)]. The expression was represented in the form of relative fold change in expression of genes under stress conditions as compared with its expression under control conditions. The R package software (R studio) was used for analysis of variance for gene expression data and different letters denote significant variation (p < 0.05).