title: Illumina MiSeq paired end sequencing
design: 16S root microbiome survey of Arabidopsis thaliana (mutant s8h (Col-0 background)) grown in a soil sample from Cologne (Germany)
library_construction_protocol: Total root DNA was extracted using the FastDNA SPIN Kit for Soil (MP Biomedicals). Samples were homogenized using the Precellys 24 homogenizer (Bertin Technologies) at 6,200 r.p.m. DNA was eluted in 80 μl of nuclease-free water and quantified using Quant-iT PicoGreen dsDNA Assay (ThermoFisher). Samples were diluted to 3.5 ng μl −1 and used in a two-step PCR amplification protocol.<br>. The V5–V7 region of the bacterial 16S rRNA gene was first amplified in triplicate reactions for each sample with primers 799F and 1192R.  Amplification was performed in a 25-μl reaction volume containing 2 U DFS-Taq DNA polymerase, 1× incomplete buffer (both Bioron), 2 mM MgCl 2 , 0.3% bovine serum albumin, 0.2 mM dNTPs (Life Technologies) and 0.3 μM forward and reverse primers. The same PCR parameters were used for each primer pair (94 °C for 2 min, 94 °C for 30 s, 55 °C for 30 s, 72 °C for 30 s and 72 °C for 10 min for 25 cycles). Single-stranded DNA and proteins were digested by adding 1 μl of Antarctic phosphatase, 1 μl Exonuclease I and 2.44 μl Antarctic Phosphatase buffer (New England Biolabs) to 20 μl of the pooled replicate reactions. Digestion was performed at 37 °C for 30 min, followed by enzyme deactivation at 85 °C for 15 min. Samples were centrifuged for 10 min at 4,000 r.p.m., and 3 μl of supernatant were used for the second PCR step in triplicate reactions to add barcodes and Illumina adapters. Reactions were prepared with barcoded primer pairs and performed as above with the number of cycles reduced to ten. PCR quality was controlled by loading 5 μl of each reaction on a 1% agarose gel. The remaining reaction volume was loaded on a 1.5% agarose gel and run at 80 V for 2 h; bands with the correct size of ∼500 base pairs were cut out and purified using the QIAquick Gel Extraction Kit (Qiagen). Concentration of the purified DNA was determined, and 30 ng DNA from each of the barcoded amplicons were pooled into one library per experiment, then purified and re-concentrated two- fold with Agencourt AMPure XP beads. Paired-end Illumina sequencing was performed in-house using the MiSeq sequencer and custom sequencing primers.