Metatranscriptomic samples from urban environments
A metagenomic-based survey of microbial (de)halogenation potential in a German forest soilhttp://www.mg-rast.org/mgmain.html?mgpage=project&project=mgp90464All MetaSUB samples were collected using Isohelix RNA buccal swabs, ensuring standardized procedures across diverse cities and sampling teams. Prior to extraction, each sample was preserved in a tube containing 400 l of RNA Shield (Zymo Research). Notably, samples stored in the DNA/RNA Shield can be frozen at temperatures of -20C to -80C for extended periods, while nucleic acids remain stable at ambient temperature (RNA up to 1 month). The collection process adhered strictly to the SOP developed collaboratively by all MetaSUB consortium members, aiming to maintain high-quality control throughout the sampling procedure. Subsequently, the swabs were extracted using the easyMag instrument (bioMrieux), following the manufacturers instructions. The input volume for extraction was 300 l, with an elution volume of 110 l. For library preparation, the total nucleic acid (TNA) from all samples underwent treatment with DNAse I (Zymo Research, Catalog # E1010), which effectively cleaves both double-stranded and single-stranded DNA. Post-DNAse digestion, the samples were subjected to the NEBNext rRNA depletion v2 (Human/Mouse/Rat) protocol, utilizing Ultra II Directional RNA (10ng) and Unique Dual Index Primer Pairs from New England Biolabs. All kits were sourced from a single manufacturer lot. Completed libraries were quantified using Qubit or equivalent methods and analyzed for size determination on a Bioanalyzer or equivalent platform. These libraries were subsequently pooled and dispatched to either the WCM Genomics Core or HudsonAlpha for final quantification using a Qubit fluorometer (ThermoFisher Scientific), TapeStation 2200 (Agilent), and qRT-PCR with the Kapa Biosystems Illumina library quantification kit. The NovaSeq 6000 (Illumina) facilitated 150-base pair paired-end sequencing at the UCSF Center for Advanced Technology. Each sequencing run included negative controls (nuclease-free water) to monitor potential laboratory and reagent contamination. Additionally, a Human Reference RNA Standard (Agilent) was incorporated for quality assurance.