On column purelink dnase treatment

Samples were homogenized in a Precellys 24 tissue homogenizer (Bertin Technologies) at 4000 rpm (skin samples) or 2500 rpm (gut samples) twice for 10 seconds. Total RNA was extracted using the Purelink RNA Mini Kit (Invitrogen™) following the manufacturer’s protocol. DNA was removed by using the On-Column Purelink DNase Treatment (Invitrogen) following the manufacturer’s protocol. Aliquots of the extracted RNA were immediately frozen at - 80°C. The concentration and quality of the RNA extract was assessed with a Nanodrop One Microvolume Spectrophotometer (Thermo Scientific™). Prior to cDNA synthesis, the RNA samples were diluted to 100 ng/µl, and a total amount of 800 ng was used for each cDNA synthesis reaction using the iScript cDNA Synthesis kit (Bio-Rad) following the manufacturer’s protocol.

Wild-type nematodes were synchronized by bleaching, plated on E. coli OP50, and maintained at 20 °C. Synchronized larval stage 4 (L4) worms were washed several times in M9 buffer and transferred to treatment bacteria or E. coli OP50. Treatments included S. maltophilia strains K279a, JCMS, and JV3. After 12 h of exposure to treatment bacteria at 25 °C, worms were collected in M9 buffer and lysed in TRIzol® (Life Technologies). 12 h of exposure to treatments was chosen because at this point bacterial accumulation in the intestine has begun [12 (link)], but almost all worms in each treatment were still alive. Only non-contaminated, un-starved populations were used for RNA extraction, and three biological replicates were collected for each treatment. Bulk RNA was extracted from these populations using PureLink RNA Mini Kit (Invitrogen), and DNase treated using On-Column PureLink® DNase Treatment (Invitrogen) following the manufacturer’s protocol. RNA quality was checked by determining 260/280 and 260/230 absorbance ratios using a NanoDrop™ 8000 Spectrophotometer and observation of 18S and 28S rRNA bands using gel electrophoresis.

Seeds from each soybean line were collected at the development stage of seeds or R7 stage [73 DAF (Day After Flowering), beginning of seed maturity] and were immediately submerged in liquid nitrogen and then stored at − 80 °C prior to RNA extraction. Seeds were ground in liquid nitrogen using a mortar and pestle, which were cooled down using liquid nitrogen after decontamination by ELIMINase (Decone Labs, Inc., King of Prussia, PA) and RNAse-free water. RNA extraction was performed using the Purelink RNA Mini Kit (Invitrogen, Carlsbad, CA). Approximately 100 mg of ground seed tissue was homogenized in 1.0 ml of lysis buffer containing 1% 2-mercaptoethanol using vortex to disperse the sample. Genomic DNA contamination was removed using the On-column Purelink DNase treatment (Invitrogen, Carlsbad, CA). The quality and quantity of RNA were assessed by the QIAxcel Advanced System and QIAxpert System Spectrophotometer (QIAGEN GmBH, Hilden, Germany), respectively. RNA samples were stored at − 80 °C until cDNA synthesis.

For Exp.1, total RNA was extracted and cDNA synthesized as previously described (Gomez de la Torre Canny et al., 2022 (link)). In brief, gut and skin samples were homogenized and total RNA was extracted using the Purelink™ RNA Mini Kit (Invitrogen™), then treated with DNase (On-Column Purelink DNase Treatment; Invitrogen), and immediately frozen at – 80°C. The iScript™ cDNA Synthesis kit (Bio-Rad) was used for cDNA synthesis with 800 ng DNase treated RNA as template, following the manufacturer’s instructions.
For samples collected in Exp.2, DNA was extracted from skin, gut and water samples using a KingFisher Flex instrument with the ZymoBIOMICS™ 96 MagBead DNA kit. First, all samples were homogenized and lysed in 750 μl lysis buffer from the kit by vortexing them horizontally in 2 ml screwcap tubes with 1.4 mm Zirconium beads for 45 min. DNA was extracted from 300 μl lysate following the kit’s protocol for the KingFisher Flex (50 μl DNAse-free water was used for elution) and samples were frozen at −20°C until examination. For a few samples we could not generate 16S rRNA gene amplicons, here, the DNA extraction was repeated using the remaining 400 μl of the lysate.