Maxwell 16 lev plant rna kit

Genes differentially expressed across different time points were selected to evaluate their expression levels through RT-qPCR for validation of RNA-Seq results. The validation was performed on biological replicates collected 24h after treatment with 2 l ha^-1 ANE in the three phenological stages. Primers were designed using the Primer-BLAST tool on NCBI (Ye et al., 2012 (link)). The list of primers is shown in Supplementary Table 2. A quantity of 3 µg total RNA extracted with a Maxwell^® 16 LEV Plant RNA Kit (Promega Corporation, USA) was converted into cDNA using a GoScript Reverse Transcription Mix, Random Primer (Promega Corporation, USA). The RT-qPCR assay was performed using a reaction mix composed of 5 μl of GoTaq qPCR Master Mix (Promega Corporation, USA), 1 μl of cDNA (4 ng μl^-1), and 0.25 μl of each gene-specific primer in a final volume of 10 μl. Three biological and two technical replicates were performed for each gene. The average Ct values of two internal reference control genes EFI1 (Solyc06g005060.2; Forward: 5’-CTGTGAGGGACATGAGGCAG-3’, reverse: 5’-CTGCACAGTTCACTTCCCCT-3’) and UBI (Solyc07g064130.1; Forward: 5’-GGACGGACGTACTCTAGCTG-3’, reverse: 5’-TCGTCTTACCCGTGAGAGTC-3’) were measured for relative expression analysis using the comparative 2^−ΔΔCt method (Schmittgen and Livak, 2008 (link)).

RNA was extracted from samples using a Maxwell 16 LEV Plant RNA kit (Promega, Madison, WI) and sequenced on an Illumina platform. Sequences were mapped to the L. starkeyi NRRL-11557 genome [24 (link)] to predict transcription start sites and quantify expression. Clustering analyses were performed in R and expression level of genes quantified with featureCounts [32 (link)] and converted into reads per kilobase million (RPKM) (Additional file 1). Samples belonging to growth, transition, or stationary phase for each bioreactor run were treated as replicates to identify genes significantly differentially expressed in MMPCS versus MMGX using DESeq2 (adjusted p value < 0.01 and log₂ fold-change > 1) [33 (link)]. Gene ontology analysis was performed with FunRich [34 (link)]. Metabolic modeling and biochemical pathway visualization utilized Cytoscape3 [35 (link)]. Promoter enrichment analysis was performed using DREME [36 (link)], Tomtom [37 (link)].

DNA was extracted using a cetyl-trimethylammonium-bromide (CTAB) based buffer (Weigel and Glazebrook, 2002).
RNA was extracted using the Maxwell 16 LEV Plant RNA Kit (Promega, Madison, WI, USA) following the manufacturer’s instructions. All samples were checked for quality on a 1.5% denaturing agarose gel.

Total RNA was extracted from whole seedlings using the Maxwell 16 LEV Plant RNA Kit (Promega). RNA was quantified using a NanoDrop (Thermo Scientific) and its integrity was analyzed by agarose gel electrophoresis. cDNA was synthetized using the cDNA Synthesis Kit (Roche). Real-time quantitative PCR was performed in a total reaction volume of 20 μL using LightCycler 480 SYBR Green I Master (Roche) and gene-specific primers (S1 Table) on a LightCycler 480 Real-Time PCR System (Roche). The normalized expression of target genes was calculated using UBC as the endogenous reference gene (S1 Table).

GLRaV-3 ORFs 2–7 and 10 were cloned into bimolecular fluorescence complementation (BiFC) binary vectors pSPYCE(M), pSPYCE(MR), pSPYNE173, and pSPYNE(R)173 [33 (link)] using primers provided in Table S1 and the same method described for the construction of the Y2H plasmids. To serve as an indicator of the success of infiltration, a red fluorescent protein-expressing plasmid pGPTVII-mCherry was constructed by cloning the mCherry gene from pmCherry (Clontech, Mountain View, CA, USA) into binary vector pGPTVII using primers and restriction enzymes provided in Table S1. Arabidopsis thaliana protein kinase CIPK24 was used as a negative control in all BiFC assays. Total RNA was extracted from A. thaliana using the Maxwell^® 16 LEV Plant RNA Kit (Promega, Madison, WI, USA). From this, the CIPK24 gene was amplified using the primers provided in Table S1, and cloned into each of the four BiFC vectors to obtain pSPYCE(M)::CIPK24, pSPYCE(MR)::CIPK24, pSPYNE173::CIPK24 and pSPYNE(R)173::CIPK24. All vectors were verified by Sanger sequencing. Also used in the BiFC assay was p19, a suppressor of gene silencing encoded by tomato bushy stunt virus cloned into the backbone pCAMBIA1300.

Stem cuttings of potato genotype SH (Bakker et al., 2003 (link)) were grown in vitro and inoculated with infective second‐stage juveniles of G. pallida population D383. Plants were kept at 18°C in the dark and nematode‐infected potato root segments were collected 3 and 6 days after infection (three biological replicates). Samples were collected and snap‐frozen in liquid nitrogen. RNA extraction was performed using the Maxwell 16 LEV‐plant RNA kit (Promega) following the manufacturer's protocol. RNA degradation and contamination were monitored on a 1% agarose gel. Purification was checked by using a NanoPhotometer spectrophotometer (IMPLEN). RNA integrity and quantification were assessed by using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies). RNA sequencing was done at Novogene by using a Novaseq 5000 PE150 platform, providing at least 50 million clean paired‐end reads of 150 bp per sample. See section 2.2 for data accessibility.