title: Metabarcoding of soil bacterial communities
design: Soil samples were taken at the termination of the experiment, that is during leaf fall in mid-October 2020. Therefore, a soil core was taken close to the stem of each tree, homogenized and stored frozen (-80C) until further processing. RNA was extracted from 250 mg soil subsamples using the Quick RNA Fecal/Soil Microbe Microprep Kit (Zymo Research) according to the manufacturers instructions. RNA was then subjected to a further clean-up step, including a DNase digestion, with Zymos RNA Clean & Concentrator-5 kit following the provided protocol. The concentration and purity of the RNA were subsequently measured spectrophotometrically (Nanodrop2000, Thermo Scientific) and 5 l RNA per sample were reverse transcribed into cDNA (iScript cDNA synthesis kit, Bio-Rad). Following the manufacturers recommendation, 2 l of cDNA were used as template for the amplification of the hypervariable V4 region of the 16S rRNA gene for prokaryotes and the ITS2 region for fungi. The prokaryote PCR mix included the primer pair 515Fm (5-GTGYCAGCMGCCGCGGTAA-3) and 806Rm {5- GGACTACNVGGGTWTCTAAT-3, \Walters, 2016 #10018}, the fungi PCR mix included the primer pair ITS86F (5- GTGAATCATCGAATCTTTGAA) and ITS4R {5- TCCTCCGCTTATTGATATGC-3, \Vancov, 2009 #11600} as well as NEBs Phusion High-Fidelity polymerase. The PCR protocols employed an initial activation step at 98 C for 30s, followed by 27 cycles consisting of 98 C for 10 s, 63 C (prokaryotes) or 60 C (fungi) for 30 s and 72 C for 30 s, and a final extension for 5 min at 72 C. To minimize PCR bias, three individual PCR reactions for each of the samples were prepared. Afterwards, all replicate samples per marker gene, tree and treatment/control were pooled together and subjected to library preparation using the Next Ultra DNA Library Prep Kit for Illumina (NEB, USA). The quality of the final samples was assessed with an Agilent Bioanalyzer 2100 system. Libraries were sequenced on an Illumina MiSeq platform, generating 2 x 250 bp paired-end reads by StarSeq GmbH, Mainz, Germany).
library_name: J14