MAG_02
The pitcher fluid from three of the 10 pitchers in each treatment group were selected from three time points (3 plants x 3 treatments x 3 timepoints (day 1, 22, 55), n=27) for simultaneous DNA/RNA extractions for metagenomic and metatranscriptomic sequencing using the Qiagen AllPrep Bacteria DNA/RNA/Protein Kit (Cat 47054), according to manufacturer directions. Briefly, flash frozen samples were thawed and pelleted, pellets were resuspended in lysis solution and bead beat for 10 minutes at 2400 RPM after which 350 L of lysate was transferred to a spin column were the DNA was collected in the column and the flow through was further processed for RNA extraction. Samples were eluted in nuclease free water and stored at -20C until quantification and library preparation. Nucleic acids were quantified using a fluorometer (Qubit, Invitrogen). Libraries were prepared for the full 27 RNA samples using NEBNext rRNA Depletion Kit (NEB#E7850) and NEBNext Ultra II Directional RNA library prep kit for Illumina (NEB#E7760) using manufacturer protocols. Metagenomic libraries (DNA) were prepared for a subset of these samples (3 plants x 3 treatments x 2 timepoints (day 1, 55), n=18) using NEBNext Ultra II FS DNA library prep kit for Illumina (NEB#78055). Sequencing was done on a NovaSeq partial lane to a sequencing depth of 55 Gb per library at Novogene Co. (Sacramento, CA USA).