Rneasy minielute cleanup kit

All flash frozen leaf samples were ground using a mortar and pestle in liquid nitrogen. RNA was extracted from individual samples using the Qiagen® RNeasy® Plant Mini Kit (Qiagen®, Germantown, MD) with some modifications to the manufacturer’s protocol. Samples were incubated at 56 °C for two minutes and vortexed occasionally to disrupt tissue. Additionally, three rounds of RPE buffer washes were done instead of two. RNA samples were DNase treated using the Ambion® TURBO DNA-free kitTM (Ambion®, Austin, TX) to remove genomic DNA contamination. DNase-treated RNA samples were cleaned with Qiagen® RNeasy® MiniElute Cleanup Kit (Qiagen®, Germantown, MD). To ensure the integrity of RNA samples, all samples were checked using an Agilent® 2100 BioanalyzerTM (Agilent®, Santa Clara, CA) and samples with RNA Integrity Number (RIN) of ≥6.9 considered to be of good quality for RNA-seq. RNA from three leaflets (obtained from three different plants) was pooled in equal amounts for each RNA pool to be analyzed. A total of 48 RNA pools (3 replicates × 2 time points × 2 treatments × 4 soybean genotypes) were submitted to the DNA facility at Iowa State University for multiplex library preparation and single end sequencing using the Illumina HiSeq 2500 instrument. Each of the 48 cDNA libraries was sequenced at a read length of 100 base pairs.

RNA was extracted from the ground collar tissue samples described above using the Qiagen® RNeasy® Plant Mini Kit (QIAGEN, Valencia, CA). Extracted RNA was DNAse treated using the Ambion® TURBO DNA-free kit™ (Invitrogen, Carlsbad, CA) to remove genomic DNA contamination. RNA clean-up was done for all DNAse-treated RNA samples using the Qiagen® RNeasy® MiniElute Cleanup Kit. RNA concentration was quantified using a NanoDrop spectrophotometer 2000 (NanoDrop Technologies, Wilmington, DE). RNA integrity was checked using the Agilent® 2100 Bioanalyzer™, and all samples were of good quality with an RNA integrity of 8 or greater. RNA samples were submitted to the DNA facility at Iowa State University for multiplex library preparation and single-end sequencing using the Illumina HiSeq 3000 instrument at a read length of 150 base pairs.

Following incubation, biofilms were washed with PBS before a cell scraper was used to dislodge the biomass, which was homogenised using a bead beater, and RNA extracted using the TRIzol™ (Life Technologies, Paisley, UK) method as described previously38 (link). Total RNA was DNase (Qiagen, Crawley, UK) treated and purified using an RNeasy MiniElute clean up kit (Qiagen, Crawley, UK), as per manufacturer’s instructions. RNA was quantified and quality assessed using a NanoDrop spectrophotometer (ND-1000, ThermoScientific, Loughborough, UK). The integrity of the RNA was assessed using an agarose gel to visualise the two distinct rRNA bands. Each isolate was grown in triplicate and a minimum of 10 μg of total RNA was submitted for each sample and sent for sequencing to The GenePool (Edinburgh, UK). RNA integrity was assessed using a Bioanalyzer where an RIN value > 7.0 was deemed acceptable for RNA-Seq using Illumina 50 base pair sequencing.

Total RNA was extracted from young fresh leaves and different stage of flowers using RNeasy Plant Mini Kit (Qiagen) following by the RNA purification by RNeasy MiniElute Cleanup Kit (Qiagen), according to the manufacture's protocol. Equal amounts of RNA from each sample were mixed together for the subsequent steps of our experiments. For mRNA library construction and deep sequencing, at least 20 µg of total RNA samples were prepared by using the TruSeq RNA Sample Preparation Kit (Illumina) for Illumina sequencing on Genome Analyzer IIx platform at CapitalBio Corporation (Beijing, China). The high quality reads obtained in this study have been deposited in the NCBI SRA database (accession number: SRA111764).