RNA-seq from sourdough synthetic microbial community
Total extracted RNA was subjected to quality control prior to library preparation. To enrich both for mRNA and other non-ribosomal transcripts, ribosomal RNAs from both eukaryotic and prokaryotic sources were depleted using Ribo-Zero rRNA removal technology. Approximately 100 ng of rRNA-depleted RNA was fragmented to an average length of 250300 bp and reverse-transcribed into double-stranded cDNA. Subsequent library preparation included end repair, A-tailing, and ligation of Illumina sequencing adapters, followed by size selection and PCR amplification, according to the manufacturers protocol. The resulting libraries were validated using the High Sensitivity DNA Kit on a Bioanalyzer 2100 instrument, and quantified using Qubit fluorometer and real-time PCR. Equimolar amounts of each library were pooled and sequenced on an Illumina NovaSeq 6000 platform, generating approximately 27 Gb of raw data per sample mate in a paired-end 150 bp (PE150) format.