Bioruptor plus ultrasonicator

Digestion of ECM-enriched pellets was performed in two steps (19 ). Samples were resuspended in 6M guanidinium hydrochloride (Gnd-HCl). Samples were reduced with 10 mM DTT for 30 min at room temperature and alkylated with 50 mM IAM for 15 min in the dark, and the reaction was quenched with additional DTT. The first digestion was done at 37 °C for 2 h with LysC (1:50 enzyme to protein ratio) in 10 mM Tris–HCl (pH 8.5) containing 2M Gnd-HCl, 2.7 M urea, and 3% acetonitrile. Prior to the 2-h incubation, samples were sonicated for 15 min (37 °C) using a Bioruptor plus ultrasonicator (Diagenode). The second digestion step was done using fresh LysC (1:50 enzyme to protein ratio) and trypsin (1:20 enzyme to protein ratio) in 600 mM Gnd-HCl, 800 mM urea, and 3% acetonitrile at 37 °C overnight. An additional sonication step was also performed prior to the overnight digest. Aliquots of digested samples containing 10 μg of protein were desalted using Pierce C18 Spin Tips (Thermo Scientific #84850) according to the manufacturer's protocol.

The methods used here are modifications of what is described in Simmons et al. (2018) (link). Roots frozen in phosphate buffer solution were thawed at 4°C and washed by sonication in a Bioruptor Plus ultrasonicator (Diagenode, Denville, NJ, United States) at 4°C for 10 min. Roots were removed from vials and rinsed twice with autoclaved water. For each plant in the subsectioning experiment, three of the clean individual roots were then cut into three sections of equal length. Roots not being processed immediately were placed in fresh sterile phosphate buffer and frozen at −80°C. Rhizosphere soil samples from the sonicated vials were centrifuged (10 min at 4 °C, 4,000 × g), and DNA was extracted by processing approximately 250 mg of each sample with MoBio’s PowerSoil kit (prior to Qiagen purchasing MoBio). DNA was extracted from root samples by grinding to a powder with liquid nitrogen, mixing 600–700 mg powder with CTAB buffer, and washing with phenol chloroform-isoamyl alcohol. For individual and sectioned roots in the subsectioning experiment, DNA was extracted using approximately 50 mg of tissue in MoBio’s PowerPlant kit. Bulk soil DNA was extracted with MoBio’s PowerSoil kit.