title: AMPLICON-Seq of rhizoplane metagenome: Vicia faba rhizoplane
design: One healthy plant from each pot was uprooted entirely at the seedling stage and shaken to remove all but approximately 1 mm of soil still attached to the roots. Next, 5 mm of the cell elongation zone was sampled from a single root of each plant. 15 root segments were collected for each plant species. To collect the rhizosphere suspensions (Rs), root segments were separately placed into micro-centrifuge tubes containing 1 mL of PBS and shaken at 300 rpm for 15 min at 4 C. Next, the same root segments were washed two times in fresh PBS, transferred to new microcentrifuge tubes containing 1 mL PBS, and then subjected to sonication using a sonication bath for 1 min at 4 C, to collect the rhizoplane suspensions (Rp). DNA was extracted from 250 mg of each soil sample and 150 L of each rhizosphere and rhizoplane suspensions using a FastPrep-24 bead-beating system and the NucleoSpin Soil Kit. The 16S rRNA gene variable regions 3-4 were amplified with primers Uni341F (5"-CCTAYGGGRBGCASCAG-3') [20] and Uni806R (5"-GGACTACHVGGGTWTCTAAT-3'). The amplicon PCR products were purified using Agencourt AMPure XP (Beckman Coulter, USA) PCR Clean-Up System (13 L AmPure beads: 20 L PCR product), normalized using SequalPrep Normalization Plate (96) Kit, concentrated using the DNA Clean and Concentrator-5 kit (Zymo Research, Irvine, CA, USA). The concentration of the pooled libraries was adjusted to 1.65 ng/L (4 nM) and sequenced on the MiSeq System with the denatured libraries adjusted to a final concentration of 16 pM and 5.0 % PhiX as internal control.