add dna extraction and library preparation assay and protocol

assays/DNAExtractionAndLibraryPreparation/protocols/DNAExtractionAndLibraryPreparationProtocol.md

+## DNA extraction and library preparation
+
+Total DNA was extracted from the aforementioned samples using the FastDNA™ SPIN Kit for Soil (MP Biomedicals, Solon, USA) or NuceloSpin Soil Mini kit for DNA from soil (Macherey-Nagel GmbH & Co. KG, Düren, Germany) following instructions from the manufacturers. DNA samples were eluted in 50 μL nuclease-free water and used for microbial community profiling. DNA samples were used in a two-step PCR amplification protocol. In the first step, V4-V7 (799F: AACMGGATTAGATACCCKG; 1192R: ACGTCATCCCCACCTTCC) of the bacterial 16S rRNA, was amplified. Under a sterile hood, each sample was amplified in triplicate in a 25 μL reaction volume containing 2 U DFS-Taq DNA polymerase, 1× incomplete buffer (Bioron GmbH, Ludwigshafen, Germany), 2 mM MgCl2, 0.3% BSA, 0.2 mM dNTPs (Life technologies GmbH, Darmstadt, Germany) and 0.3 μM forward and reverse primers. PCR was performed using the following parameters: 94 °C/2 min, 94 °C/30 s, 55 °C/30 s, 72 °C/30 s, 72 °C/10 min for 25 cycles. Afterwards, 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 GmbH, Frankfurt, Germany) to 20 μl of the pooled PCR product. Samples were incubated at 37 °C for 30 min and enzymes were deactivated at 85 °C for 15 min. Samples were centrifuged for 10 min at 3,000 × g and 3 μL of this reaction were used for a second PCR, prepared in the same way as described above using the same protocol but with cycles reduced to 10 and with primers including barcodes and Illumina adapters (Thiergart, T. et al., 2020). PCR quality was controlled by loading 5 μL of each reaction on a 1% agarose gel and affirming that no band was detected within the negative control. Amplicon concentration was determined fluorescently (Quant-iT™ PicoGreen™, Invitrogen), and equivalent DNA amounts of each of the barcoded amplicons were pooled in one library. Then, 80 μL of the pooled library was loaded in a 1.5% agarose gel and run for 2 h at 80 V. Subsequently, bands with a size of ∼500 bp were cut out and purified using the QIAquick gel extraction kit (Qiagen, Hilden, Germany). The final library concentration was estimated fluorescently (Quantus™ Fluorometer, Promega). Paired-end Illumina sequencing was performed in-house using the MiSeq sequencer and custom sequencing primers at the Max Planck Institute for Plant Breeding Research.
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