RNAseq of Geobacter sulfurreducens: anode at -0.15 V, N2 fixing, replicate 1.
Anodes from MECs were removed and placed directly in RNAprotect Bacteria Reagent (QIAGEN) to preserve total RNA. The anode was vigorously scraped using a sterile razor to remove the biofilm cells, which were deposited in the preserving reagent for 5 min. After centrifuging the samples (5,000 g for 10 min at 4 C), the supernatant was removed, and the pellets were stored at 80 C until RNA was extracted. RNA extraction was performed using the RNeasy Mini Kit (QIAGEN) following the manufacturers instructions. Lysozyme and proteinase K were used to lyse bacterial cells, and DNAse I was added to remove DNA contamination. The RNA samples were subjected to ribodepletion, where ribosomal RNA (rRNA) was removed from the samples using the RiboMinus Transcriptome Isolation kit for bacteria followed by the RiboMinus Concentration Module (Thermo Fisher Scientific, Waltham, MA). cDNA libraries for next-generation sequencing were prepared using the NEBNext Ultra RNA library prep kit for Illumina (New England BioLabs Inc., Ipswich, MA).