RNA-Seq of bacterial 16S rRNA amplicons: generated from marine sediment
The a 16S metagenomic amplicon library compatible with the Illumina MiSeq System has been prepared basically as described before (Bhnke et al., 2019; Perner et al., 2022). Briefly summarized, RNA was extract from around 300 mg sediment sample using the NucleoBond RNA Soil Mini kit (Macherey-Nagel, Dren, Germany) followed by subsequent DNaseI digestion using the RapidOut DNA Removal kit (Thermo Fisher Scientific GmbH, Waltham, Massachusetts, USA). Total environmental RNA was then transcribed into cDNA with Invitrogens SuperScript VILOTM cDNA Synthesis Kit (Life TechnologiesTM, Darmstadt, Germany). Purified cDNA (Zymo Clean & Concentrator-5, Irvine, CA, USA) was then used to generate 16S ribosomal amplicons for the bacterial hypervariable V3 and V4 regions using the primer pairs S-D-Bac-0341-b-S-17 and S-D-Bac-0785-a-A-21 as described by Klindworth et al., (2013). Sequencing was done in collaboration with the Competence Centre for Genomic Analysis (CCGA) of the Institute of Clinical Molecular Biology (IKMB, CAU Kiel, Germany) in a 2x300 bp paired-end sequencing run on the Illumina MiSeq platform (Illumina, St. Diego, USA).