MiSeq of bacteria fungi and protists from bulk soil after potato harvest
Total DNA and RNA were extracted simultaneously following a protocol optimised for 2 g soil (Harkes et al., 2019a). This extraction method comprises bead beating, precipitation of humic acids with an ammonium aluminium sulphate solution, and phenol-chloroform extraction. cDNA was synthesised from the extracted RNA using a Maxima First Strand cDNA Synthesis Kit for RT-PCR (Fermentas, Thermo Fisher Scientific Inc., USA) following the manufacturers instructions. In preparation for the first step of the library construction, all DNA and cDNA samples were diluted to 1 ng l-1 and 0.1 ng l-1, respectively. Adhering to the procedure described in (Harkes et al., 2019a), the library was generated following a two-step PCR procedure. The first step consisted of the amplification of organismal group-specific 16S and 18S rRNA regions. To this end, locus-specific primers extended with an Illumina read area and an appropriate adapter were employed that targeted the V4 region of 16S of bacteria, and the V9, V7-V8, V5-V7 of 18S of protozoa, fungi and metazoa, respectively (Table 2). For PCR amplification, 3 l of the diluted samples were used as templates. PCR was carried out with the following temperature profile: 3 s at 95 C, followed by 39  (10 s at 95 C; 20 s at 55 C; 20 s at 72 C) and a final extension step of 5 s at 72 C. The first PCR step was conducted in triplicate for all the samples and for all four organismal groups. The second PCR step was performed on 40x diluted amplicons of the first PCR products. The second PCR aimed at attaching the sample-specific Illumina index combination and the Illumina sequencing adapter to the amplicons of the first PCR. The following temperature profile was employed: 3 s at 95C, followed by 10 (10 s at 95C, 30 s at 60C, 30 s at 72C) and a final extension step of 5 s at 72C. A random selection of products of the first and second PCR steps was checked on gel to ensure that the amplifications were successful in producing amplicons of the expected size. Lastly, PCR products were pooled together and sent out for sequencing to Useq (Utrecht Sequencing Facility, Utrecht, The Netherlands). Illumina MiSeq sequencing was performed using a 2 x 300 bp V3 kit.