title: Ro32-DNA-T0-plt4
design: 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 (indicated as RNA) 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, ITS 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 and V7-V8 of 18S of protozoa and  fungi,  respectively, and ITS2 of fungi.  After the initial amplification, the PCR amplicons were diluted 40 folds with Milli-Q water and the secodn aplicication  was conducted with Illumina sequencing adaptors and index sequences for sample multiplexing. Lastly, PCR products were pooled together and sent out for sequencing to Useq  (Utrecht Sequencing Facility, Utrecht, The Netherlands) in three batches (see BioSample metadata). IlluminaNovaSeq sequencing was performed NovaSeq SP 2 kit  (2250bp).   The sequences submitted here  contain amplicons of 4 primers, to demultiplex use the following primer combinations:  515F/806R (Caporaso et al., 2012) targeting the V4 region of 16S (bacteria) - FW: GTGCCAGCMGCCGCGGTAA, RV: GGACTACHVGGGTWTCTAAT ,  1391F/EukBr (Medlin et al., 1988; Lane, 1991) to amplify the V9 region of 18S (protists)  - FW:GTACACACCGCCCGTC, RV: TGATCCTTCTGCAGGTTCACCTAC FF 390.1 /FR1 (Verbruggen et al., 2012) targeting the V7-V8 region of 18S for fungi - FW: CGWTAACGAACGAGACCT, RV: AICCATTCAATCGGTAIT gITS7/ITS4ngs (Tedersoo et al., 2018) targeting the ITS2 region of fungi - FW: GTGARTCATCGARTCTTTG, RV: TCCTSCGCTTATTGATATGC  Part of the methodology (and pat of the data) is used and explained explained in Cazzaniga et al. 2023 - Differential effects of cover crops on the soil microbiome under various densities of the plant-parasitic nematode Meloidogyne chitwoodi.