add microbiota reconstruction experiment assay and protocol

assays/Multi-speciesMicrobiotaReconstitutionExperiments/protocols/Multi-speciesMicrobiotaReconstitutionExperimentsProtocol.md

+## Multi-species microbiota reconstitution experiments
+
+We used the gnotobiotic FlowPot system (Duran, P. et al. , 2018), (Kremer, J. M. et al., 2018) to grow *At* and *Lj* plants with and without bacterial SynComs. In brief, the system allows for even inoculation of each growth pot with microbes by the flushing of pots with the help of a syringe attached to the bottom opening. Sterilized seeds are placed on the matrix (peat and vermiculite, 2:1 ratio), and pots are incubated under short-day conditions (10 h light, 21 °C; 14 h dark, 19 °C), standing in customized metal racks in sterile plastic boxes with filter lids (SacO2 microboxes, www.saco2.com). For SynCom preparation, bacterial commensals were grown separately in liquid culture for 2–5 d to reach high density, harvested and washed in 10 mM MgSO4. Equivalent amounts of each strain were combined to yield the desired SynComs with an optical density (OD600) of 1. An aliquot of 200 µl of the SynCom as reference sample for the experiment start, and aliquots of 50 µl of the individual strains were taken and stored at −80 °C for sequencing. The SynCom was added to the desired medium to reach a final OD600 of 0.02. FlowPots were each flushed with 50 ml of inoculum (medium/SynCom mix). Generally, the medium used for inoculation was 0.25× B&D (Broughton, W. J. & Dilworth, M. J., 1971) supplemented with 1 mM KNO3 for both plant species. In experiments D, F, K and M (Supplementary Table 2), 0.5× MS (2.22 g l−1 Murashige+Skoog basal salts, Duchefa; 0.5 g l−1 MES anhydrous, BioChemica; adjusted to pH 5.7 with KOH) was used for *Arabidopsis*. The two plant species were grown in separate FlowPots side-by-side, with ten pots in total per plastic box. After 5 weeks of growth, roots were harvested and cleaned thoroughly from attached soil using sterile water and forceps. Lotus root segments containing nodules were omitted. Soil samples from planted and unplanted pots were collected as rhizosphere and soil samples, respectively. All root (epiphytic and endophytic compartments), rhizosphere and soil samples were transferred to Lysing Matrix E tubes (FastDNA Spin Kit for Soil, MP Biomedicals), frozen in liquid nitrogen and stored at −80 °C for further processing. DNA was isolated from those samples using the FastDNA Spin Kit for Soil, and from individual strains of the SynCom via quick alkaline lysis (Bai, Y. et al., 2015), then subjected to bacterial community profiling or absolute quantification of bacteria. For RNA isolation, samples were harvested the same way and processed using the RNeasy Plant Mini kit (Qiagen).
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