title: 16s Ribosomal RNA gene sequencing of root associated bacterial communities from Allium fistulolium grown in grassland soil
design: Sampling whole rootstocks followed protocols established to reduce the presence of plastid DNA (Ikeda et.al, 1990) Soil was removed by shaking and washing the rootstock in an excess volume of sterile H2O. Rootstocks were weighed and homogenised in 120ml of pre-chilled bacterial cell enrichment (BCE) buffer (1L H2O, 50 ml 1M Tris, 10ml Triton X100, 2 ml ?-mercaptoethanol) using a blender at high speed for three minutes. The homogenate was filtered through a layer of sterilized Miracloth and centrifuged at 1,800 rpm for 5 min at 10C. The supernatant was further centrifuged at 4,000 rpm for 20 min and the pellet re-suspended in 5ml of BCE buffer.  Samples were filtered into one 15 ml falcon tube through 4 layers of sterilized Kimwipe. Filtrates were then centrifuged at 4,000 rpm for 10 min at 10C. The supernatant discarded and the pellet re-suspended in 10 ml of 50 mM Tris?HCl (pH7.5). The filtration and spin was repeated and the pellet resuspended in 6 ml of 50 mM Tris?HCl. This bacterial cell suspension was over-laid on 5 ml of 50% (w/v) Iodixanol-Tris?HCl solution and centrifuged at 4,000 rpm for 40 min at 10C. Approximately 0.5 ml bacterial cell fraction was collected with a sterile glass capillary dropper and diluted with an equal volume of sterile water and centrifuged at 10,000 rpm for 1 min. The pellet was then resuspended in 350l sterile water and an aliquot of 20l immediately plated onto L-Broth agar plates prior to storing at -80C. Bacterial pellets were resuspended in 200?l TE buffer (Tris-HCl, 10 mM; EDTA, 1 mM; pH 7.0) for 30mins. The DNA was extracted with N-acetyl-N-trimethyl ammonium bromide (CTAB)/NaCl. DNA was precipitated overnight at -20C with 70% ethanol before purification by phenol/chloroform/isoamyl 25:24:1 and chloroform/isoamyl 24:1 cleaning. The final DNA precipitate was dried and resuspended in 35?l TE buffer and stored frozen (-20C), after the protocol recommended by the DOE Joint Genome Institute (JGI) for isolation of genomic DNA from bacteria (http://my.jgi.doe.gov/general/protocols.html).DNA extracts were quality checked using agarose gel electrophoresis and quantified using a spectrophotometer (Nanodrop ND-1000; NanoDrop Tech. Inc). Samples containing a sufficient yield of high mass and purity DNA were selected, at approximately 50ng of DNA template per sample. The V4 variable region of the 16S rRNA gene was amplified using primers F515 (5?-NNNNNNNNGTGTGCCAGCMGCCGCGGTAA?3?) and R806 (5??GGACTACHVGGGTWTCTAAT?3?) [78], including a unique 12bp index sequence in each of 24 reverse primers to allow samples to be multiplexed on a single sequencing run. Primers also included sequencing adapters to allow the products to be directly sequenced on Illumina platforms, and were supplied in the NEXTflex 16S V4 Amplicon Sequencing Kit (Bioo Scientific). PCR conditions consisted of an initial 98C for 2min denaturation followed by 30 cycles of 98C for 45s, 50C for 60s, and 72C for 90s, and a final extension of 72C for 10min. PCR clean-up used a bead based clean-up and size selection step (Agencourt XP Ampure beads). The quality of the final products was quantified by gel electrophoresis and Qubit fluorometer (Life Technologies). The samples were diluted to 1ng/?l and pooled in a 4nM library and sequenced using the 250bp paired-end protocol on an Illumina MiSeq.