RNA-SIP of diffuse vent fluids at Axial Seamount: Anemone in 2014
Diffuse vent fluid samples were incubated for specific times with isotopically-labeled bicarbonate to target microbial autotrophs. This technique, RNA-stable isotope probing, results in the RNA of active autotrophs being isotopically heavier than those that are inactive. The incubations were filtered, and RNA was extracted following the protocol in the mirVana miRNA isolation kit (Ambion). Biological material was removed from the filter pieces with a bead-beating step using RNA PowerSoil beads (MoBio, Carlsbad, CA, USA), followed by an extraction by acid phenol:chloroform. A total volume of 100L RNA was extracted and any remaining DNA was removed using the Turbo-DNase kit (Ambion). RNA was purified, concentrated using the RNAeasy MinElute kit (Qiagen, Hilden, Germany). RNA was then sheared to a fragment size of 175 bp (2013 and 2014) using a Covaris S-series sonicator. The Ovation Complete Prokaryotic RNA-Seq DR multiplex system (Nugen) was used for removal of ribosomal RNA, cDNA synthesis, and metatranscriptomic library preparation